JP4460228B2 - New method for measuring nucleic acids - Google Patents

Description

【０２１５】
なお、表１の各塩基配列を有するオリゴヌクレオチドは、nec1遺伝子のどこの領域にハイブリダイズ（対応）するかを示したのが図１である。
又、NECM-Rの塩基配列は、表中の塩基配列１１、１２、１３、及び１５番目（５’末端から）の塩基が標的核酸（遺伝子）の配列に対して変異したものである（下記の表２参照）。同様にＮＥＣＭ−Ｆの塩基配列は、表中の１１、１３、１４及び１５番目（５’末端から）の塩基が変異したものである（下記の表２参照）。
【０２１６】

【０２１７】
操作手順は以下の通りである。
（１）Streptomyces turgidiscabies IFO16080のゲノムDNA（IFOより購入）を鋳型として、ａとｄのプライマーを一つのセットとして、及びｂとｃのプライマーを一つのセットとして用い、それぞれについてＰＣＲを行った。なお、ゲノムＤＮＡは下記のようにして調製した。
（２）その産物について、アガロースゲル電気泳動を行った。そして増幅核酸に相当するバンドをそれぞれ切り出し、増幅核酸を抽出した。
（３）各抽出物を混合し、新たなプライマーを添加せずに、ＰＣＲを行った。
（４）その産物について、前記２）と同様にして電気泳動を行った。ＡからＢまでの長さの産物に相当するバンドのみを切り出し、増幅産物を抽出した。
（５）抽出物を鋳型として、ａとｂのプライマーをセットとして用い、ＰＣＲを行った。
（６）その産物について、前記２）と同様にして電気泳動を行った。ＡからＢまでの長さに相当するバンドのみを再び切り出した。
（７）シーケンサーにて変異挿入を確認した。又、ＤＮＡ量を定量した。
【０２１８】
６）標的核酸プローブ及び内部標準核酸プローブの作製
表２に示されるNECB-24は標的核酸プローブの塩基配列であり、NECMB-24は、内部標準核酸プローブのものである。本実施例においては、標的核酸プローブをNECB-24と、内部標準核酸プローブをNECMB-24と呼称する。
NECB-24は、前記に記載する方法にて５’末端をBODIPY FLで標識し、NECMB-24は、前記に記載する方法にて５’末端を6-TAMRAで標識した。双方とも３’末端の３’ＯＨ基をリン酸化して用いた。
表中、NECB-24の１０、１１、１２及び１４番目の塩基C、T、G及びAが、NECMB-24においては、おのおのA、G、C及びTに変異したものである。
【０２１９】
７）ＰＣＲは次の条件で行った。
・変性反応：95℃、15sec
・アニーリング及び検出：62℃、18sec
・伸長（extention）：72℃、14sec
・Taqポリメラーゼ：Gene Taq（日本ジーン株式会社）
・プライマー添加濃度：500nM、前記ｅ及びｆ（図１及び表１参照）
・プローブ添加濃度：50nM
・鋳型核酸（Template）：内部標準核酸NECM1及び標的核酸NECB1のPCR断片（ａ及びｂプラマーをセットにしてPCR増幅産物：約700bp）の混合液
・測定装置：スマートサイクラーシステム（タカラバイオ株式会社）（以下、便宜上、スマートサイクラーという。）。
【０２２０】
・使用チャンネル：
チャンネル１(Ex、450〜495nm;Em、505〜537nm)。
チャンネル３(Ex、527〜555nm;Em、565〜605nm)。
チャンネル１でBODIPY FLの蛍光強度測定を行い、同時にチャンネル３で6-TAMRAの蛍光強度測定を行った。
【０２２１】
８）土壌からのＤＮＡの抽出
Bio101キット（土壌用）を用いて土壌から標的核酸を抽出して、更にＷizard DNA Clean-Up systemを用いて精製したものを土壌サンプル（試料）とした。
【０２２２】
Ｂ）実験例
１）作製プローブの評価
標的核酸プローブNECB-24及び内部標準核酸プローブNECMB-24の対応核酸（標的核酸又は内部標準核酸）とハイブリダイズしたときの蛍光強度変化（減少）率と解離曲線を作成した。反応用バッファーはＰＣＲ反応で用いるものと同じものを使用した。
その結果を図２及び３に示した。両プローブとも最大蛍光強度変化率は約７０％であり、まずまずの変化率を示した。NECB-24は標的核酸と内部標準核酸とのＴｍ値の差は、２０℃弱であり、６２℃付近で検出すれば、内部標準核酸を検出せずに標的核酸のみを検出できることが明らかとなった。又、同様にNECMB-24は内部標準核酸と標的核酸とのＴｍ値の差は、２０℃弱であり、６２℃付近で検出すれば、標的核酸を検出せずに標的核酸のみを検出できることが明らかとなった。それで、両プローブともＰＣＲの増幅産物の検出は、６２℃で行なうことにした。
【０２２３】
２）プローブの特異性
前記Ａ）の７）に記載のＰＣＲ反応条件にて、NECB-24を核酸プローブ、プライマーとして前記ａ、ｂを使用して、標的核酸のＰＣＲを行なった際のリアルタイムモニタリングした結果を図４に示した。又、同様にNECMB-24を核酸プローブ、プライマーとして、前記ｃ、ｄを使用して、内部標準核酸のＰＣＲを行なった際のリアルタイムモニタリングした結果を図５に示した。両プローブとも約３０％との高い蛍光強度変化率（蛍光消光率）を示すことが分かった。又、両プローブともに、標的核酸と内部標準核酸の混合比に応じ、得られる蛍光強度変化率が変化した。この結果より、本方法により、定量的に標的核酸の定量が行えるものと考えられた。
【０２２４】
３）ＤＮＡ濃度と蛍光強度変化率の関係式の作成
種々のＤＮＡ濃度における蛍光強度変化率を前記両プローブで測定した。その結果を図６Ａ及び６Ｂに示した（この図の作製に当たり、前記の本発明のデータ解析方法の記載の処理を行い蛍光強度変化率を求めた。）。
【０２２５】
各図から、未知試料中の標的核酸の濃度若しくはコピー数を求める方法について以下に記す。先ず、標的核酸を含む未知試料にコピー数の判明している内部標準核酸、及び標的核酸プローブ並びに内部標準核酸プローブを添加して、ＰＣＲを行う。双方の核酸の増幅が反応系の蛍光強度変化から目視可能となったとき、双方の核酸の蛍光強度変化率を求める。当該蛍光強度変化率を図６Ａ及び６Ｂに当てはめて、当該蛍光変化率を示すときの増幅された内部標準核酸量（Ａとする。）及び標的核酸量（Ｂとする。）を求める。内部標準核酸量の核酸増幅前のコピー数をＡ’とすると、標的核酸の増幅前のコピー数Ｂ’とする。Ｂ’は次式で求めることが出来る：
Ｂ’＝（Ｂ／Ａ）Ａ’
【０２２６】
４）従来の検量線を用いたスマートサイクラーによる標的核酸の定量
既知濃度の標的遺伝子サンプルに、実際に土壌試料から抽出した核酸抽出試料を、濃度を変えて混合し、正確に定量を行なえるかどうか検討した。、内部標準核酸プローブと標的核酸プローブは、双方とも、反応チューブに添加し、それぞれ内部標準核酸と標的核酸を同時に検出した。
【０２２７】
先ず、既知濃度の外部標準遺伝子（nec1遺伝子）により検量線を作成した（図７Ａ、７Ｂ）。土壌から抽出した核酸を含む試料０μｌ、１μｌ、２μｌ、３μｌに、６，６９０，０００コピーの標的遺伝子（nec1遺伝子）を添加したものを鋳型とし、本サンプル中のnec1遺伝子の定量を、前記の検量線を用いて行なった。その結果、入れる土壌試料の量が多くなるに応じて、グラフが右にシフトすること、つまり増幅が遅れて起こることが明らかになった（図８）。この遅れによりにＣｔ値が上昇し、その結果、定量値は実際に添加した標的遺伝子（nec1遺伝子）よりも低く算出される事が分かった（表−３参照）。以上の結果より、従来の外部標準遺伝子による検量線を用いた既存法（リアルタイム定量的ＰＣＲ法）では、ＰＣＲ阻害物質を含むサンプルについて、標的遺伝子の定量を正確に行えないことが明らかとなった。
【０２２８】

【０２２９】
５）本発明方法を用いたスマートサイクラーによる標的核酸の定量
本発明の内部標準核酸を添加した方法により、標的核酸の増幅前のコピー数を求めた。表３に方法の相違による定量値の相違を示した。その結果、入れる土壌試料の量が多くなるに応じてグラフが右にシフトするのは前記と同様であるが、本発明では、増幅効率の低下（増幅の遅れ）が観察されたが、内部標準遺伝子の増幅も同様に低下したため、添加した標的遺伝子量と本発明方法による定量値とがほぼ一致することが明らかになった。
【０２３０】
以上の結果より、内部標準核酸を添加し、標的核酸及び内部標準核酸由来の蛍光強度の変化量若しくは変化率から、標的核酸の濃度若しくはコピー数を求める本発明の新規測定方法の有用性が明らかになった。又、本発明の方法の標準偏差値は、従来のリアルタイム定量的ＰＣＲ法のそれより小さく、定量値のぶれが小さいことが明らかとなった。これらの結果から、ＰＣＲ阻害物質が存在する場合は勿論のこと、存在しない場合においても、その正確性において従来法に対する本発明の優位性が証明された。
【０２３１】
６）モデル系で本発明方法の実施
前記実験例を踏まえて、標的核酸と内部標準核酸の添加比を変化させ、それぞれのＰＣＲの増幅産物を前記二種のプローブの蛍光強度変化量をリアルタイムモニタリングした。その結果、添加比に応じて蛍光強度変化量が得られることを確認した。
【０２３２】
７）実際の土壌試料についての定量
前記実験例を踏まえて、標的遺伝子（nec1遺伝子）のコピー数が未知である土壌試料（標的核酸を含む。）について、既知量の内部標準核酸を用いて定量した。
土壌抽出核酸試料１μｌ及び２μｌに、内部標準核酸を６６．９コピーほど測定系に添加し、標的遺伝子（nec1遺伝子）の定量を本発明の方法にて行った。その結果、土壌抽出核酸試料を１μｌ添加した系では、６７．５コピーと定量された。又、土壌抽出核酸試料を２μｌ添加した系の定量値は１４２．５コピーであった。本定量値は、土壌抽出核酸試料を１μｌ添加した系の約２倍であることから、ほぼ正確に土壌抽出核酸試料中の標的遺伝子（nec1遺伝子）を定量出来ているものと判断される。
【０２３３】
実施例２
Mergneyらのプローブを用いて、実施例１と同様の検討を行った。
１）プローブの合成
プローブの合成は、（株）日本遺伝子研究所（http://www.ngrl.co.jp/）によって合成されたものを使用した。
２）内部標準核酸（ＤＮＡ遺伝子）
実施例１と同様である。
【０２３４】
３）標的核酸プローブ及び内部標準核酸プローブの作製
表４に示されるNECB-24 acceptorの塩基配列は実施例１に記載の標的核酸プローブの塩基配列であり、NECMB-24 acceptorの塩基配列は、前記同様に実施例１に記載の内部標準核酸プローブのものである。本実施例においては、標的核酸プローブをNECB-24 acceptorと、内部標準核酸プローブをNECMB-24 acceptorと呼称する。NECB-24 acceptorは、５’末端をLCRed640と呼ばれる蛍光色素で標識したもの、NECMB-24 acceptorは、５’末端をLCRed705と呼ばれる蛍光色素で標識したものである。双方とも３’末端の３’ＯＨ基をリン酸化した。上記の２種のプローブに修飾した蛍光色素（LCRed705、LCRed640）は、ＦＲＥＴ現象のアクセプター色素として機能する。これら色素を励起するために必要なドナー色素を修飾したプローブが、Nec-donoｒである（配列は表４参照）。当該プローブの３’末端はＦＩＴＣにして修飾されており、それぞれの標的遺伝子が存在した場合、色素標識部位が互いに向き合うように、２塩基間隔を置いてハイブリダイズするように設計されている（図９参照）。この様に、Nec-donorとNECB-24 acceptorあるいは、Nec-donorとNECMB-24 acceptorが隣接してハイブリダイズした場合、ＦＲＥＴ現象が発生し、蛍光キャラクターが大きく変化する。この変化量から、それぞれのプローブセットの対応核酸を検出することが出来る。
又、当該プローブの塩基配列は、NECMB-24 acceptorにおいては、実施例１で用いたNECMB-24と同様であり、NECB-24 acceptorにおいては、実施例１で用いたNECB-24と同様である。
【０２３５】

【０２３６】
４）ＰＣＲは、以下に記述した以外の条件は、実施例１と同様の条件で行った。・各プローブ（NECB-24 acceptor、NECBM-24 acceptor、Nec-donor）添加濃度：200nM
・測定装置：ライトサイクラーシステム（ロッシュ株式会社）（以下、便宜上、ライトサイクラーという。）。
・使用した蛍光測定用チャンネル
チャンネル２（Ex、470〜490nm;Em、640nm）。
チャンネル３（Ex、470〜490nm;Em、710nm)。
チャンネル２でLCRed640の測定を行い、同時にチャンネル３でLCRed705の蛍光測定を行った。
５）土壌からのＤＮＡの抽出
実施例１と同様の方法で実施した。
【０２３７】
Ｂ）実験例
１）作製プローブの評価
標的核酸プローブ（Nec-donorとNECB-24 acceptorのセット）及び内部標準核酸プローブ（Nec-donorとNECMB-24 acceptorのセット）の対応核酸（標的核酸又は内部標準核酸）とハイブリダイズしたときのアクセプター蛍光変化率と解離曲線を作成することにより作製プローブの評価を行った。反応用バッファーはＰＣＲ反応で用いるものと同じものを使用した。
【０２３８】
その結果を図１０及び１１に示した。両プローブともアクセプター蛍光強度増加化率は約４０％であり、顕著にアクセプターの蛍光強度が増加した。実施例１で使用したNECB-24及びNECMB-24と同様、完全相補的な核酸（標的核酸と標的核酸プローブ、又、内部標準核酸と内部標準核酸プローブ）とミスマッチを有する核酸とのＴｍ値の差は、２０℃弱であり、６２℃付近で検出すれば、ミスマッチを有する核酸（標的核酸と内部標準核酸プローブ、又、内部標準核酸と標的核酸プローブ）を検出せずに完全相補的な核酸のみを検出できることが明らかとなった。以上の結果より、実施例１と同様、両プローブともＰＣＲの増幅産物の検出は、６２℃で行なうことにした。
【０２３９】
２）標的核酸と内部標準核酸の濃度比に対する標的核酸由来の蛍光強度増加率と内部標準核酸由来の蛍光強度増加率との関係式の作成
標的核酸（塩基配列と添加する濃度が判明しているので、標準核酸というべきであるが、本発明においては、便宜上、標的核酸と称することにした。）と内部標準核酸（濃度比を様々に変化させたもの）を鋳型として、前記実施例１のＡ）の７）に記載の反応条件にて、ＰＣＲを実施し、標的核酸由来の増幅産物と内部標準核酸由来の増幅産物とを、それぞれMergneyらのプローブにてリアルタイムモニタリングした。その結果から、標的核酸の増幅産物由来の蛍光強度増加率（NECB-24 acceptorプローブの蛍光強度増加率）と内部標準核酸の増幅産物由来の蛍光強度増加率（NECMB-24 acceptorプローブの蛍光強度増加率）との比を求め、それをＰＣＲ前の標的核酸と内部標準核酸の濃度比との関係を求めた（図１２参照）。その結果、ＰＣＲ前の標的核酸と内部標準核酸の濃度比と、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率との比との間に高い相関があることが明らかとなった。従って、本関係式からＰＣＲ前の標的核酸と内部標準核酸の濃度比を知ることが出来ることが示された。尚、この図の作製に当たり、前記の本発明のデータ解析方法の記載の処理を行い、蛍光強度変化率を求めた。
【０２４０】
図１２から、未知試料中の標的核酸の濃度若しくはコピー数を求める方法について以下に記す。先ず、標的核酸を含む未知試料にコピー数の判明している内部標準核酸、及び標的核酸プローブ並びに内部標準核酸プローブを添加して、ＰＣＲを行う。双方の核酸の増幅が反応系の蛍光強度変化から目視可能となったとき、双方の核酸の増幅産物由来の蛍光強度増加率を求める。当該蛍光強度増加率から、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率を求める。この比を図１２に当てはめて、標的核酸（Ｂ）と内部標準核酸（Ａ）の比（Ｂ／Ａ）を求める。内部標準核酸量の核酸増幅前のコピー数（Ａ’）は既知であるので、標的核酸の増幅前のコピー数（Ｂ’）は次式で求めることが出来る：
Ｂ’＝（Ｂ／Ａ）Ａ’
【０２４１】
３）従来方法による外部標的（標準）核酸（遺伝子）の検量線を用いたライトサイクラーによる標的核酸の定量
Mergneyらのプローブを用いた以外、実施例１と同様のサンプル、同様の条件にて、既知濃度の標的遺伝子サンプルに、実際に土壌試料から抽出した核酸抽出試料（以下、土壌抽出核酸抽試料という場合がある。）を、濃度を変えて混合し、正確に定量を行なえるかどうか検討した。なお、使用する土壌は、標的遺伝子であるNec1遺伝子を含まないことを予め確認して用いた。又、内部標準核酸の検出用プローブ（NECMB-24 acceptor）と標的核酸プローブ（NECB-24 acceptor）及びNec-donorは、双方とも、反応チューブに添加し、それぞれ内部標準核酸と標的核酸を同時に検出した。
【０２４２】
先ず、土壌から抽出した核酸を含む試料３μｌに6.690,000コピーのNec1遺伝子を添加し、nec1遺伝子の定量を行なった。又、土壌抽出核酸試料を添加せず、同数のnec1遺伝子を添加したサンプルについても、nec1遺伝子の定量を行った。その結果、実施例１と同様、土壌抽出核酸試料を添加したものは、土壌抽出核酸試料を添加しなかったサンプルに比べて、増幅が遅れて起こることが明らかとなった。この結果より、土壌抽出核酸試料には、増幅効率を低下させるＰＣＲ阻害物質が含まれていることが示唆された。この遅れによりに、Ｃｔ値が大きくなり、その結果、定量値は低く算出される事が分かった（表５参照）。以上の結果より、外部標的核酸（遺伝子）の検量線を用いた従来方法（リアルタイム定量的ＰＣＲ方法）では、ＰＣＲ阻害物質を含むサンプルについては正確に標的遺伝子の定量を行えないことが明らかとなった。
【０２４３】
４）本発明方法を用いたライトサイクラーによる標的核酸の定量
本発明の内部標準核酸を添加した方法を、Mergneyらのプローブを用いて実施し、標的核酸の増幅前のコピー数を求めた。表５に方法の相違による測定値の相違を示した。その結果、土壌抽出核酸試料を添加したサンプルでは、前記の従来方法（リアルタイム定量的ＰＣＲ法）と同様、増幅効率の低下（増幅反応の遅れ）が観察されたが、内部標準遺伝子の増幅効率も同様に低下したため、測定値が一定となることが明らかになった。この結果より、内部標準核酸を添加し、標的核酸及び内部標準核酸由来の蛍光強度の変化量若しくは変化率から、標的核酸の濃度若しくはコピー数を求める本発明の新規測定方法が、Mergneyらのプローブを用いても有効であることが明らかになった。
なお、表５の各方法の数値は、分析回数を５回ずつ行って得た数値の平均値である。
【０２４４】

【０２４５】
５）実際の土壌試料についての測定
前記実験例を踏まえて、標的遺伝子（nec1遺伝子）のコピー数が未知である土壌試料について、既知量の内部標準核酸を用いて当該コピー数を測定した。使用した土壌サンプルは、実施例１の実際の土壌試料についての測定で用いたもの（土壌抽出溶液１μｌ当たり６７．５コピーの標的遺伝子（nec1遺伝子）が存在すると測定されたサンプル）と同様である。
【０２４６】
実施例１と同様、土壌抽出核酸試料１μｌ及び２μｌに、内部標準核酸を６６．９コピーほど測定系に添加し、標的核酸（遺伝子）（nec1遺伝子）の測定を本発明の方法にて行った。その結果、土壌抽出核酸試料を１μｌ添加した系では、８３．２コピーと測定された。当該測定値は、実施例１の測定値（６７．５コピー）とほぼ同様の測定値を示した。又、土壌抽出核酸試料を２μｌ添加した系の測定値は１５２．７コピーであった。当該測定値は、土壌抽出核酸試料を１μｌ添加した系の約２倍であることから、ほぼ正確に土壌抽出核酸試料中の標的遺伝子（nec1遺伝子）を測定出来ているものと判断される。以上の結果より、Mergneyらのプローブを用いても、土壌中の標的遺伝子を正確に測定できることが強く示唆された。
【０２４７】
実施例３
分子ビーコン（Molecular beacon)プローブ（前記の公知発明方法の（３）に記載）を用いて、実施例１、２と同様の検討を行った。
１）プローブの合成
プローブは、エスペックオリゴサービス株式会社(http://www.business-zone.com/espec-oligo/)によって合成されたものを使用した。
【０２４８】
２）標的核酸プローブ及び内部標準核酸プローブ
表６に示されるNECB-24 beaconの塩基配列は標的核酸プローブの塩基配列であり、NECMB-24 beaconの塩基配列は、内部標準核酸プローブのものである。NECB-24 beaconは、ステム・ループ構造をとるよう４塩基ずつ塩基が付加されており、５’末端は、FAMと呼ばれる蛍光色素で、３’末端はDABCYLと呼ばれるクエンチャー物質で、それぞれ標識されている。NECMB-24 beaconも、ステム・ループ構造をとるよう４塩基ずつ塩基が付加されており、５’末端は、６−TAMRAと呼ばれる蛍光色素で、３’末端はDABCYLと呼ばれるクエンチャー物質で、それぞれ標識されている。
プローブ配列は表６に示したとおりである。
【０２４９】

【０２５０】
３）ＰＣＲは、以下に記述した以外の条件は、実施例２と同様の条件で行った。
・各プローブ添加濃度：200nM
・測定装置：スマートサイクラーシステム（タカラバイオ株式会社）（以下、便宜上、スマートサイクラーという。）。
・使用チャンネル：
チャンネル１(Ex、450〜495nm;Em、505〜537nm)。
チャンネル３(Ex、527〜555nm;Em、565〜605nm）。
チャンネル１でNECB-24 beaconに標識したFAMの蛍光測定を行い、同時にチャンネル３でNECB-24 beaconに標識した6-TAMRAの蛍光測定を行った。
【０２５１】
４）土壌からのＤＮＡの抽出
実施例１と同様の方法で実施した。
【０２５２】
Ｂ）実験例
１）作製プローブの評価
標的核酸プローブ（NECB-24 beacon）及び内部標準核酸プローブ（NECMB-24 beacon)の対応核酸（標的核酸又は内部標準核酸）とハイブリダイズしたときの蛍光強度増加率と解離曲線を作成した。反応用バッファーは前記同様にＰＣＲ反応で用いるものと同じものを使用した。
【０２５３】
その結果を図１３及び１４に示した。完全相補的な核酸を添加した場合、両プローブとも蛍光強度が約２，０００％増加した。又、ミスマッチを有する核酸を添加した場合、両プローブとも、ほとんど蛍光強度の増加が見られなかった。この結果より、両プローブともミスマッチを有する核酸とはほどんどハイブリダイズしない（標的核酸と内部標準核酸プローブとが、また内部標準核酸と標的核酸プローブとがハイブリダイズしない）と判断された。以上の結果より、実施例１と同様、両プローブともＰＣＲの増幅産物の検出は、６２℃で行なうことにした。
【０２５４】
２）標的核酸と内部標準核酸の濃度比に対する標的核酸由来の蛍光強度増加率と内部標準核酸由来の蛍光強度増加率との関係式の作成
標的核酸と内部標準核酸の濃度比に対する標的核酸由来の蛍光強度増加率と内部標準核酸由来の蛍光強度増加率との関係式を実施例２と同様の方法で求めた。未知試料中の標的核酸の濃度若しくはコピー数を求める方法も、実施例２と同様である。
【０２５５】
３）Molecular beaconをプローブとして用いた際の、外部標準遺伝子の検量線を用いた従来方法による標的核酸の測定結果と、本発明の方法による標的核酸の測定結果との比較
Molecular beaconプローブを用いる以外、実施例１、２と同様に測定した。サンプル、同様の条件にて、外部標準遺伝子の検量線を用いた従来方法による標的核酸の測定結果と、本発明の方法による標的核酸の測定結果との比較を行った。その結果、土壌抽出核酸試料を添加した供試サンプルでは、実施例１、２と同様、増幅効率の低下（増幅反応の遅れ）が観察されたが、添加した標的核酸（遺伝子）を正確に測定可能であった（表７参照）。この結果より、molecular beaconをプローブとして用いても、本発明の新規測定方法を、好適に実施可能であることが明らかになった。
【０２５６】

【０２５７】
実施例４
前記の本願発明方法Ｂのｉ）〜ｉｖ）の（１）〜（３）の特質を有する核酸プローブを利用する方法を適用する実施例である。
１）標的核酸及び内部標準核酸
実施例１と同様にした。
２）標的核酸プローブの合成
前記実施例１に記載のNECB-24と同じ塩基配列とした（表２参照）。そして、５’末端シチジル酸に蛍光物質テキサスレッド（Texas Red）を、５’末端から６番目のチミンにクエンチャー物質Dabcylを鎖中標識した。
当該核酸プローブの合成はエスペックオリゴサービス社に委託した。
【０２５８】
３）内部標準核酸プローブの合成
前記実施例１に記載のNECMB-24と同じ塩基配列とし、５’末端シチジル酸に蛍光物質Alexa 594を、５’末端から６番目のチミンにクエンチャー物質Dabcylを鎖中標識した。調製は標的核酸プローブと同様に委託合成した。
【０２５９】
４）標的核酸と標的核酸プローブ、及び内部標準核酸と内部標準核酸プローブのハイブリダイゼーション条件：
実施例１と同様にした。
５）測定条件
励起波長及び測定蛍光波長は以下の通りである。
・標的核酸プローブ：励起波長：590nm(4nm幅）；測定蛍光波長：610nm(4nm幅）。
・内部標準核酸プローブ：励起波長：560nm(4nm幅）；測定蛍光波長：580nm(4nm幅）。
【０２６０】
６）実際の土壌核酸抽出試料中の標的遺伝子（nec1遺伝子）の測定
土壌核酸抽出試料中の標的遺伝子（nec1遺伝子）を、前記の本願発明方法Ｂのｉ）〜iv）の（１）〜（３）の特質を有する核酸プローブを利用して、実施例１と同様の内部標準核酸を添加するＰＣＲ法を介した遺伝子測定方法を実施して、測定した。ＰＣＲ条件等は実施例１と同様である。その結果、土壌核酸抽出試料１μＬ中に含まれる標的遺伝子（nec1遺伝子）は６３．２コピー、土壌核酸抽出試料２μＬ中に含まれる標的遺伝子（nec1遺伝子）は１４８．１コピーであった。この測定値は、実施例１〜３で得られた結果とほぼ同等であることから、本願発明方法Ｂのｉ）〜iv）の（１）〜（３）の特質を有する核酸プローブを用いても、本発明の新規測定方法を、好適に実施可能であることが示された。
【０２６１】
前記したように実施例１〜４において、種類の異なる核酸プローブを用いて、共通の標的遺伝子（nec1遺伝子）を、同様の方法（内部標準核酸を添加する手法）で測定した。その結果、ほぼ同様の良好な結果を得ることができた。遺伝子増幅過程をリアルタイムモニタリング出来るプローブであれば、プローブの種類に関わらず、本発明の新規測定方法に適応可能であることが明らかとなった。
【０２６２】
実施例５
前記公知方法のＦＲＥＴ現象を利用する場合ののｖ）〜ｉｘ）に記載のヘヤーピンプローブをプライマー（サンライズプライマー）として用い、リアルタイム定量的ＰＣＲを行って、標的核酸の核酸増幅前のコピー数を求める方法に本発明方法を適用する実施例である。
１）標的核酸の塩基配列
実施例１〜３で使用したNec1遺伝子
２）内部標準核酸塩基配列
実施例１〜３で使用したNec1遺伝子に対応する内部標準遺伝子
【０２６３】
３）プライマー
（１）標的核酸検出用フォーワードプライマー
・塩基配列：acagtta CTCCATGAACTGTACCGCGACCAG（アンダーライン：付加した配列）
・５’末端をドナー色素FAMで標識し、３’末端から１２塩基目のＴをクエンチャー色素 DABCYLで標識した。
（２）内部標準核酸検出用フォーワードプライマー
・塩基配列：agctttg CTCCATGAAagcTtCCGCGACCAG（アンダーライン：付加した配列、小文字：標的核酸検出用フォーワードプライマーと配列が異なる部位。つまり、標的核酸と内部標準核酸の配列が異なる部位）
・５’末端のリン基をドナー色素６−TAMRAで標識し、３’末端から１２塩基目のＴを前記同様にクエンチャー色素DABCYLで標識した。
（３）リバースプライマー
・塩基配列：前記の表１のｆ（NECS-R）
【０２６４】
前記のフォワードプライマー（標的核酸検出用フォーワードプライマー及び内部標準核酸検出用フォーワードプライマー）は、サンライズプライマーとして機能するよう設計されており、遺伝子増幅によりプライマー内の分子内２次構造が解消されることで、蛍光発光するように設計されている。従って、本蛍光発光を測定することで、増幅産物をリアルタイムモニタリングすることが可能となる。又、各フォワードプライマーは、内部標準遺伝子と標的遺伝子の塩基配列の異なる部位を認識し、それぞれ特異的に結合し、伸長するように設計されている。このため、各フォーワードプライマー（標的核酸検出用フォーワードプライマー及び内部標準核酸検出用フォーワードプライマー）によって、標的核酸あるいは内部標準核酸を特異的に増幅することが出来る。又、標的核酸検出用フォーワードプライマー及び内部標準核酸検出用フォーワードプライマーは、それぞれ異なる蛍光色素で標識されているため、標的核酸由来の増幅増幅産物と内部標準核酸由来の増幅産物を、同一反応系内で同時に検出することが出来る。
前記プライマーは、エスペックオリゴサービス株式会社(http://www.business-zone.com/espec-oligo/)に委託し、合成した。
【０２６５】
４）ＰＣＲは次の条件で行った。
・変性反応：95℃、15sec
・アニーリング及び検出：55℃、5sec
・伸長（extention):72℃、10sec
・Taqポリメラーゼ：Gene Taq（日本ジーン株式会社）
・プライマー添加濃度：100nM
・鋳型核酸（Template)：内部標準核酸NECM1及び標的核酸NEC1のＰＣＲ断片（ａ及びｂプラマーをセットにしてＰＣＲ増幅産物：約７００ｂｐ）の混合液
・測定装置：スマートサイクラーシステム（タカラバイオ株式会社）
【０２６６】
・使用チャンネル：
チャンネル１（Ex、450〜495nm;Em、505〜537nm)。
チャンネル３(Ex、527〜555nm;Em、565〜605nm)。
チャンネル１で標的核酸検出用フォーワードプライマーに標識したFAMの測定を行い、同時にチャンネル３で内部標準核酸検出用フォーワードプライマーに標識した6-TAMRAの蛍光測定を行った。
【０２６７】
５）標的核酸と内部標準核酸の濃度比に対する標的核酸由来の蛍光強度増加率と内部標準核酸由来の蛍光強度増加率との関係式の作成
標的核酸と内部標準核酸の濃度比を、様々に変化させたものを鋳型として、前記実施例２の４）に記載の反応条件にて、ＰＣＲを実施し、標的核酸由来の増幅産物と内部標準核酸由来の増幅産物とを、それぞれリアルタイムモニタリングした。その結果から、標的核酸の増幅産物由来の蛍光強度増加率（NECB-24 acceptorプローブの蛍光強度増加率）と内部標準核酸の増幅産物由来の蛍光強度増加率（NECMB-24 acceptorプローブの蛍光強度増加率）との比を求め、それをＰＣＲ前の標的核酸と内部標準核酸の濃度比との関係を求めた（図１５参照）。その結果、ＰＣＲ前の標的核酸と内部標準核酸の濃度比と、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率との比との間に高い相関があることが明らかとなった。従って、当該関係式からＰＣＲ前の標的核酸と内部標的核酸の濃度比を知ることが出来ることが示された。以上の結果から、サンライズプライマーを用いた方法に於いても、実施例２，３の場合と同様に、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率との比から、ＰＣＲ前の標的核酸と内部標準核酸の濃度比を求めることが可能であることが明らかとなった。
【０２６８】
４）従来方法の検量線を用いたスマートサイクラーによる標的核酸の測定
既知濃度の標的遺伝子サンプルに、実際に土壌試料から抽出した核酸抽出試料を、濃度を変えて混合し、正確に測定を行なえるかどうか検討した。内部標準核酸用プライマーと標的核酸用プライマーは、双方とも、反応チューブに添加し、それぞれ内部標準核酸と標的核酸を同時に検出した。
【０２６９】
先ず、既知濃度の外部標的遺伝子（nec1遺伝子）による検量線を作成した。土壌から抽出した核酸を含む試料３μｌに、6,690,000コピーの標的遺伝子（nec1遺伝子）を添加したものを鋳型とし、本サンプル中のnec1遺伝子の測定を、前記の検量線を用いて行なった。又、土壌抽出核酸試料を添加せず、同数のnec1遺伝子を添加したサンプルについても、nec1遺伝子の測定を行った。その結果、実施例１〜３の場合と同様、土壌抽出核酸試料を添加することで、土壌抽出核酸試料を添加しなかったサンプルに比べて、グラフが右にシフトすること、つまり増幅反応が遅れて起こることが明らかになった。この遅れによりにＣｔ値が大きくなり、その結果、測定値は実際に添加した標的核酸（遺伝子）（nec1遺伝子）よりも低く算出される事が分かった（表８参照）。以上の結果より、外部標的核酸（遺伝子）による検量線を用いた従来方法（リアルタイム定量的ＰＣＲ法）では、サンライズプライマーを用いた場合に於いても、ＰＣＲ阻害物質を含むサンプル中の標的遺伝子の測定を、正確に実施できないことが明らかとなった。
【０２７０】

【０２７１】
５）本発明方法を用いたスマートサイクラーによる標的核酸の測定
本発明の内部標準核酸を添加した方法により、標的核酸の増幅前のコピー数を求めた（表８参照）。その結果、添加する土壌試料の量が多くなるに応じてグラフが右にシフトするのは前記と同様であるが、本発明では、増幅効率の低下（増幅反応の遅れ）が観察されたが、内部標準遺伝子の増幅も同様に低下したため、添加した標的遺伝子量と本発明方法による測定値とがほぼ一致することが明らかになった。
【０２７２】
以上の結果より、反応系に標的核酸に対応する内部標準核酸を添加し、標的核酸及び内部標準核酸由来の蛍光強度の変化量若しくは変化率を、サンライズプライマーを用いて求め、標的核酸の濃度若しくはコピー数を求める本発明の新規測定方法の有用性が明らかになった。
又、前記（実施例）と同様の検討を、前記本発明方法Ｂのｉｘ）に標記したハイブリダイゼーションにより蛍光が減少する核酸プローブを、プライマーとして用いた場合にも、上記と全く同様の傾向を示す結果が得られた（data not shown)。この結果より、遺伝子増幅過程をリアルタイムモニタリング出来るプライマーであれば、プライマーの種類に関わらず、本発明の新規測定方法に適応可能であることが明らかとなった。
【０２７３】
実施例６
遺伝子増幅法をＰＣＲ方法からLAMP(Loop-Mediated Isothermal Amplification)方法（一つの核酸増幅方法）に替えて、実施例１〜４までと同様の検討を行った。
１）標的遺伝子
実施例１〜５までと同様、nec1遺伝子とした。
２）土壌からのＤＮＡの抽出
実施例１と同様の方法で実施した。
３）プローブの合成
プローブ及びプライマーは、エスペックオリゴサービス株式会社(http://www.business-zone.com/espec-oligo/）に委託製造した。４）内部標準核酸（ＤＮＡ遺伝子）の作製
実施例８として後述する人工遺伝子の取得法にて作製した。
５）LAMP用プライマーの設計と配列
プライマーの設計は、栄研化学株式会社(http://www.eiken.co.jp/)のホームページ上の、LAMP法用のプライマー設計ソフトにて行った。
本プライマーは、標的核酸と内部標準核酸に完全に相補的な配列を有しており、標的核酸と内部標準核酸を同時に増幅可能なプライマーとなっている。
【０２７４】

【０２７５】
６）標的核酸プローブ及び内部標準核酸プローブ
表９に示されるNECB-23 LAMPは、LAMP法で増幅される標的核酸由来の増幅産物を検出するためのプローブの塩基配列であり、NECMB-23 LAMPは、LAMPで増幅される内部標準由来の増幅産物を検出するための核酸プローブである。本実施例においては、標的核酸プローブをNECB-24と、内部標準核酸プローブをNECMB-24と呼称する。
【０２７６】
NECB-24は、前記に記載する方法にて５’末端をBODIPY FLで標識し、NECMB-24は、前記に記載する方法にて５’末端を6-TAMRAで標識してある。双方とも３’末端の３’ＯＨ基をリン酸化して用いた。
本プローブは、実施例１で用いた、対応核酸とハイブリダイズすることで蛍光強度が減少するタイプの核酸プローブである。
表中、NECB-23 LAMPの９、１０、１１、１２及び１４番目の塩基t、a、c及びtが、NECMB-23 LAMPにおいては、おのおのA、T、G及びAに変化したものである。
【０２７７】
Ｂ）実験例
１）作製プローブの評価
標的核酸プローブ（NECB-23 LAMP）及び内部標準核酸プローブ（NECMB-23 LAMP）の対応核酸（標的核酸又は内部標準核酸）とハイブリダイズしたときの蛍光強度変化率｛消光（減少率）率｝と解離曲線を作成した。反応用バッファーはLAMP法で用いるものと同じものを使用した。
【０２７８】
その結果を図１６及び１７に示した。NECB-23 LAMPは標的核酸と内部標準核酸とのＴｍ値の差は、約２０℃強であり、６０〜７０℃の範囲で検出すれば、内部標準核酸を検出せずに標的核酸のみを良好に検出できることが明らかとなった。又、同様にNECMB-23 LAMPは内部標準核酸と標的核酸とのＴｍ値の差も、NECB-23 LAMPと同様、約２０℃強であり、６０〜７０℃の範囲で検出すれば、標的核酸を検出せずに標的核酸のみを良好に検出できることが明らかとなった。以上の結果より、ＰＣＲの増幅産物の検出は両プローブとも、LAMP法の反応温度である６５℃で行なうこととした。
【０２７９】
２）LAMP法による遺伝子増幅は次の条件で行った。
・反応温度：65℃、60min
・ポリメラーゼ，dNTP等を含む増幅キット：Loopamp DNA増幅試薬キット（栄研化学株式会社）
・プライマー添加濃度：800nM(NEC FIP、NEC BIP)、200nM(NEC F3、NEC B3)(表10参照)
・プローブ添加濃度：各200nM
・鋳型核酸（Template）：内部標準核酸NECM1及び標的核酸NEC1のＰＣＲ断片（表１のａ及びｂプラマーをセットにしてＰＣＲ増幅産物：約７００ｂｐ）の混合液
・測定装置：スマートサイクラーシステム（タカラバイオ株式会社）（以下、便宜上、スマートサイクラーという。）。
【０２８０】
・使用チャンネル：
チャンネル１（Ex、450〜495nm;Em、505〜537nm)。
チャンネル３（Ex、527〜555nm;Em、565〜605nm)。
チャンネル１でBODIPY FLの測定を行い、同時にチャンネル３で6-TAMRAの蛍光測定を行った。
【０２８１】

【０２８２】
３）標的核酸と内部標準核酸の濃度比に対する標的核酸由来の蛍光強度増加率と内部標準核酸由来の蛍光強度増加率との関係式の作成
標的核酸と内部標準核酸の濃度比を、様々に変化させたものを鋳型として、前記２）に記載の反応条件にて、ＬＡＭＰ反応を実施し、標的核酸由来の増幅産物と内部標準核酸由来の増幅産物とを、それぞれ表１０に示した核酸プローブにてリアルタイムモニタリングした。その結果から、標的核酸の増幅産物由来の蛍光強度変化｛消光（減少）率｝（以下、単に蛍光消光率という。）（NECB-23 LAMPの蛍光消光率）と内部標準核酸の増幅産物由来の蛍光消光率（NECMB-23 LAMPの蛍光消光率）との比を求め、それをＬＡＭＰ反応前の標的核酸と内部標準核酸の濃度比との関係を求めた。その結果、実施例２〜４で示したＰＣＲの場合と同様、増幅前の標的核酸と内部標準核酸の濃度比と、標的核酸の増幅産物由来の蛍光消光率と内部標準核酸の増幅産物由来の蛍光消光率との比との間に高い相関が見られた(data not shown)。従って、ＰＣＲ方法の場合と同じように、本関係式から遺伝子増幅前の標的核酸と内部標準核酸の濃度比を知ることが出来ることが示された。
【０２８３】
４）LAMP法介した土壌中の標的遺伝子（nec1遺伝子）の測定
前記実験例を踏まえて、内部標準核酸を用い、かつLAMP法介した本発明の新規遺伝子測定手法にて、標的遺伝子（nec1遺伝子）のコピー数が未知である土壌抽出核酸試料（標的核酸を含む。）中に存在する標的核酸量を測定した。
実験は、実施例１〜５のＰＣＲを介した手法と同様、土壌抽出核酸試料１μｌ及び２μｌに、内部標準核酸を６６．９コピーほど測定系に添加し、標的遺伝子（nec1遺伝子）の測定を、LAMP法介した遺伝子測定方法にて行った。土壌抽出核酸試料は実施例１〜５と同様のものを用いた。その結果、土壌抽出核酸試料を１μｌ添加した系では、７９．６コピーと測定された。又、土壌抽出核酸試料を２μｌ添加した系の測定値は１３９．２コピーであった。本測定値は、実施例１〜５で得られた結果とほぼ同等であることから、正確に土壌抽出核酸試料中の標的遺伝子（nec1遺伝子）を測定出来ているものと判断される。
以上の結果より、内部標準核酸を添加し、標的核酸及び内部標準核酸を核酸プローブにて検出することを特徴とする本発明の新規核酸（遺伝子）測定方法に、ＰＣＲ方法以外の遺伝子増幅法を適応可能であることが、証明された。
【０２８４】
実施例７
Streptomyces turgidiscabiesのゲノムＤＮＡから、nec1遺伝子の分離・濃縮を行うことが必要な場合は、以下の方法で分離・濃縮した。
使用する制限酵素は、Bfa1、BsaJ1、BssK1、Dde1、Msel、Msp1を使用した（全て、New England BIOLABSから購入した。）。これらは、２本鎖ＤＮＡの特定の４塩基の配列を認識して、切断する制限酵素であるため、確率論上、２本鎖ＤＮＡは約４０塩基の長さに切断されると考えられる（任意の４塩基の配列は、２５６通りあるため、１種の４塩基認識の制限酵素で切断される部位は、２５６塩基毎に出現すると考えられる。今回は６種の制限酵素で切断するため、２５６÷６＝４２．６となり、平均して約４０塩基毎に切断されると考えられる。）。しかしながら、nec1遺伝子は、上記６種の制限酵素を用いて処理することにより、nec1 遺伝子は５３６塩基の長さとなり、比較的長い２本鎖ＤＮＡとして存在する。このため、ＤＮＡの長さを指標として簡便に標的遺伝子を含む画分を回収可能となる。
【０２８５】
上記６種の制限酵素を各１μｌ、Mse1に付属のx10 bufferを１０μｌ、ジャガイモそうか病が発生した土壌から前記実施例１と同様にして抽出した核酸溶液（８０μｌ）を混合し、３７℃で８時間反応させた後、更に６０℃で８時間反応させた。nec1この断片を含む画分を、フィルター｛マイクロコン−１００（分割分子量＝１００，０００）、ミリポア｝に通した。１００ｂｐ以下のＤＮＡのみが本フィルターを通過するため、５３６塩基のnec1遺伝子は、フィルター上に残留する。nec1フィルター上に残留したnec1遺伝子を含む画分を回収し、乾燥後、１μｌのミリポア純水に再溶解した。
【０２８６】
当該再溶解中のnec1遺伝子を実施例１の実験例７）と同様の手順にて測定した。又、濃縮の有無を確認するために制限酵素処理前の核酸溶液についても前記同様の方法でnec1遺伝子を測定した。核酸溶液の添加量は、それぞれ１０μｌとした。その結果、濃縮処理を実施した核酸溶液中のnec1遺伝子数は４７９０コピー／μｌ、濃縮を実施しない場合の核酸溶液中の当該遺伝子数は６３コピー／μｌであった。以上の結果より、本方法により、簡便に標的核酸（nec1遺伝子）の濃縮が可能であることが証明された。
【０２８７】
実施例８
任意の塩基配列を有する長鎖の人工遺伝子の合成は、以下の方法で実施した。
１）一本鎖オリゴヌクレオチドＤＮＡの合成
合成する一本鎖オリゴヌクレオチド遺伝子は、実施例６で用いたnec1遺伝子に対応する内部標準遺伝子とした。その配列を以下に示す。塩基数は２７４ｂｐであり、小文字とアンダーラインで示した部分が、標的核酸であるｎｅｃ１遺伝子と配列の異なる部分である。当該一本鎖オリゴヌクレオチドＤＮＡは、エスペックオリゴサービス株式会社に委託し、合成した。
【０２８８】
合成されたオリゴＤＮＡは電気泳動にかけ、目的の一本鎖オリゴＤＮＡ遺伝子（２７４ｂｐ）由来のバンドが得られたかを確認したが、２７０ｂｐ付近に明瞭なバンドは確認できなかった。又、全体に電気泳動パターンはスメアであり、２７０ｂｐ以外についても、明瞭なバンドは確認されなかった。以上の結果は、様々な長さの一本鎖オリゴＤＮＡが合成されており、目的の一本鎖オリゴＤＮＡ遺伝子の割合は、非常に低いということを示唆していると考えられる。以上の結果より、通常のオリゴＤＮＡを合成する手法では、任意の配列を有する長鎖の遺伝子は得ることは不可能であることが強く示唆された。
【０２８９】

【０２９０】
２）一本鎖オリゴヌクレオチドＤＮＡを鋳型としたＰＣＲ反応
目的のＤＮＡを得るため、上記の一本鎖オリゴヌクレオチドＤＮＡ遺伝子を鋳型として、ＰＣＲ増幅を行った。使用したプライマーの配列を表１１に示した。ＰＣＲの条件は、以下に示した通りである。
・変性反応：95℃、60sec
・アニーリング及び検出：62℃、60sec
・伸長(extention):72℃、60sec
・サイクル数：40サイクル
・Taqポリメラーゼ：Gene Taq（日本ジーン株式会社）
・プライマー添加濃度：各500nM
・ＰＣＲ装置：ｉCycler（バイオラッド社製）
【０２９１】

【０２９２】
ＰＣＲ反応終了後、電気泳動を行った。しかしながら、２７０ｂｐ付近に明瞭なバンドは確認されなかった。そこで、得られたＰＣＲ産物を１００倍に希釈し、これを鋳型として再度、同条件でＰＣＲを実施し、電気泳動で産物を確認した。その結果、僅かに２７０ｂｐ付近にバンドが確認できた。しかしながら、泳動パターンが明瞭でなく全体にスメアであったため、再度、ＰＣＲ産物を１００倍に希釈したものを鋳型としてＰＣＲを実施し、電気泳動で産物を確認したところ、２７０ｂｐ付近に明瞭なバンドが確認された。実際に、目的の産物が得られた事を確認するため、ＰＣＲ産物の塩基配列を決定した。以下にその工程を示す。
【０２９３】
ＰＣＲ産物は、マイクロスピンS-400HRカラム（アマシャムファルマシア社製）を用いて精製した。シークエンシング反応は、ダイデオキシターミネーター・シークエンシングキット（アプライドバイオシステムズ社製）を用いて実施し、得られた産物は、自動塩基配列決定装置（ABI PRISM TM 377、アプライドバイオシステムズ社製）を用いて、塩基配列を決定した。その結果、前記のＰＣＲ産物は、目的の内部標準遺伝子の配列を有していることが確認できた。
以上の結果より、本発明方法により任意の配列を有する長鎖の人工遺伝子を、簡便に合成可能であることが証明された。
【０２９４】
実施例９
前記本願発明方法Ｂの核酸プローブに標識する色素の種類について調べた。下記の塩基配列を有するデオキシリボポリヌクレオチドを核酸プローブとして、下記の塩基配列を有するデオキシリボポリヌクレオチドを、それに対応する核酸とした。なお、各デオキシリボポリヌクレオチドは前記の核酸合成機を用いて調製した。
【０２９５】
・標的核酸プローブ用：5'CCCCCCCCCTTTTTT3'
・標的核酸用：5'AAAAAAGGGGGGGGGGGG3'
標的核酸プローブ用のデオキシリボポリヌクレオチドに色素を標識するには、前記実施例１と同様な方法を用いて行った。
蛍光測定は下記の条件で行った。
【０２９６】
（１）ハイブリダイゼーション溶液のコンポーネント
・合成ＤＮＡ 320nM（終濃度）
・核酸プローブ 80nM（終濃度）
・ＮａＣｌ 50mM（終濃度）
・ＭｇＣｌ₂ 1mM（終濃度）
・トリス−塩酸緩衝液（pH＝7.2） 100mM（終濃度）
・ミリＱ純水 1.6992ml
・終全量 2.0000ml
（２）ハイブリダイゼーションの温度：51℃
（３）測定条件：
励起光 ：表９に記載
測定蛍光色：表９に記載
【０２９７】
その結果を、表９に示した。表から分かるように、本発明の蛍光色素の蛍光強度を減少させるために用いる蛍光色素として、前記記載の中でも好適なものは、蛍光強度の減少率が１５％以上のものなどを挙げることができる。
【０２９８】
実施例１０
前記Morrisonらの方法を適用する実施例（二種の核酸プローブを用いる方法である。そして、当該核酸プローブが標的核酸にハイブリダイズする前は、当該プローブは互いにハイブリダイズしているものである。）である。
１）標的核酸及び内部標準核酸
実施例１と同様にした。
２）標的核酸プローブの合成
標的核酸プローブは次の二本のオリゴヌクレオチドからなるものである。
ａ） 5'-TCCATGAACT GTACCGCGACーCAG-3'（実施例１の標的核酸プローブNECB-24の５’末端のシチジル酸を削除した塩基配列）
ｂ） 5'-TCGCGGTACA GTTCATGGA-3'（前記ａ）にハイブリダイズ塩基配列）
ａ）は、５’末端のリン酸基のＯＨ基に蛍光物質6-TAMRAを標識した。
ｂ）は、３’末端のアデノシンのデオキシリボースの３’位ＣのＯＨ基にクエンチャー色素Dabcylを標識した。
【０２９９】
３）内部標準核酸プローブ
標的核酸と同様に、前記Morrisonらの方法に用いられているプローブである。
次の塩基配列を二本のオリゴヌクレオチドからなるものである。
ａ）5'-TCCATGAAAGCTTCCGCGACCAG-3’（実施例１の標的核酸プローブNECMB-24の５’末端を塩基削除したもの）
ｂ）5'-TCGAA GTTCATGGA-3'（前記ａ）にハイブリダイズ塩基配列）
ａ）は、５’末端のリン酸基のＯＨ基に蛍光物質FAMを標識した。
ｂ）は、３’末端のアデノシンのデオキシリボースの３’位ＣのＯＨ基にクエンチャー色素Dabcylを標識した。
【０３００】
４）標的核酸プローブ、内部標的核酸プローブの調製方法
実施例４と同様に行った。
５）標的核酸と標的核酸プローブ、及び内部標準核酸と内部標準核酸プローブのハイブリダイゼーション条件：
実施例４と同様にした。
【０３０１】
６）測定条件
励起波長及び測定蛍光波長は以下の通りである。
・標的核酸プローブ：励起波長：527〜555nm；測定蛍光波長：565〜605nm。
・内部標準核酸プローブ：励起波長：450〜495nm；測定蛍光波長：505〜537nm。
【０３０２】
７）土壌サンプル（ハイブリダイゼーション反応の阻害物質）を含ない測定系でのハイブリダイゼーション反応の蛍光強度測定
標的核酸及び内部標準核酸の濃度を種々替えて、ハイブリダイゼーション前後の蛍光強度変化量を測定し、標的核酸及び内部標準核酸の検量線を作成した。
標的核酸プローブの濃度：500nM
内部標準核酸プローブの濃度：500nM
【０３０３】
８）土壌サンプル
標的核酸を含まない土壌について実施例１と同様に調製した。なお、標的核酸を含まないことを、実施例１の方法にて確認した。
９）土壌サンプル（ハイブリダイゼーション反応の阻害物質）を含む測定系での標的核酸の回収試験標的核酸の測定
（１）土壌サンプルを測定系１００μLに１００μL宛を添加して、測定系を複雑系にした。更に標的核酸を２．５×１０⁸コピー／１０μLになるように添加した。内部標準核酸の濃度を種々変えて、蛍光強度を測定してた。その結果、内部標準核酸についての検量線が得られた。内部標準核酸の検量線についての変化率を計算し、標的核酸にあてはめた。その結果、複雑系における標的核酸の検量線を得た（実際は、簡便化法によって行った。内部標準核酸についての二つの検量線の勾配（傾き）を求め、同一濃度での二つの検量線の差を求めた。そして、内部標準核酸についての複雑系の検量線の勾配と、同一濃度での二つの検量線の差をそのまま、標的核酸について当てはめた。）。
【０３０４】
（２）土壌サンプル中の標的核酸のコピー数。
測定結果は、２．８×１０⁸コピー／１０μL、標的核酸の回収率は１１２％であった。
【０３０５】
【発明の効果】
本発明の核酸の新規測定方法は前記のように構成されているので、標的核酸と標的核酸プローブのハイブリダイゼーション反応及び／又は核酸増幅反応を阻害する物質、又は多型核酸が含まれている試料においても、標的核酸を微量で、しかも正確かつ簡便、短時間に特異的に測定できる。しかも複数の標的核酸を同時に測定できる。
【図面の簡単な説明】
【図１】遺伝子（標的核酸）にハイブリダイズする各プライマーの当該遺伝子におけるハイブリダイゼーションの位置。
【図２】標的核酸（遺伝子（wild type)）又は内部標準核酸（nec1変異型遺伝子（mutated type））と標的核酸プローブ（NECB-24）とのハイブリダイゼーション物（標的核酸プローブ複合体）の解離曲線。
【図３】標的核酸（nec1遺伝子（wild type））又は内部標準核酸（nec1変異型遺伝子（mutated type））と内部標準核酸プローブ（NECMB-24）とのハイブリダイゼーション物（標的核酸プローブ複合体）の解離曲線。
【図４】標的核酸プローブによる標的核酸と内部標準核酸との混合物を鋳型とするＰＣＲのリアルタイムモニタリング。
【図５】内部標準核酸プローブによる標的核酸と内部標準核酸との混合物を鋳型とするＰＣＲのリアルタイムモニタリング。
【図６】ＤＮＡ濃度と標的核酸プローブに標識された色素の蛍光強度変化量（率）の関係式を示す図。
Ａ：標的核酸
Ｂ：内部標準核酸
【図７】Ａ：土壌試料が添加されていない測定系における各種ＤＮＡ濃度におけるＰＣＲのリアルタイムモニタリグ。
Ｂ：Ａに基づく検量線
【図８】従来公知の定量的ＰＣＲ方法によるリアルタイムモニタリング。鋳型核酸：nec1遺伝子のａ及びｂプローブをセットするＰＣＲ産物７００万
【図９】 ハイブリダイゼーションした際のプローブの位置関係
【図１０】 Nec-donorとNECB-24 acceptorのプローブセットの内部標準遺伝子とnec1遺伝子に対する解離曲線。
【図１１】 Nec-donorとNECMB-24 acceptorのプローブセットの内部標準遺伝子とnec1遺伝子に対する解離曲線。
【図１２】 増幅前の標的核酸と内部標準核酸の濃度比に対する、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率との関係式
【図１３】 NECMB-24 beaconの内部標準遺伝子とnec1遺伝子に対する解離曲線。
【図１４】 NECMB-24 beaconの内部標準遺伝子とnec1遺伝子に対する解離曲線。
【図１５】 増幅前の標的核酸と内部標準核酸の濃度比に対する、標的核酸の増幅産物由来の蛍光強度増加率と内部標準核酸の増幅産物由来の蛍光強度増加率との関係式（サンライズプライマーを使用した場合）
【図１６】 NECB-23 LAMPの内部標準遺伝子とnec1遺伝子に対する解離曲線。
【図１７】 NECB-23 LAMPの内部標準遺伝子とnec1遺伝子に対する解離曲線。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye.
Specifically, in this method, a substance that inhibits a nucleic acid hybridization reaction and / or a nucleic acid amplification reaction corresponds to a target nucleic acid probe and a target nucleic acid that specifically hybridize to the target nucleic acid in an existing measurement system or reaction system. In this method, an internal standard nucleic acid probe that specifically hybridizes to the internal standard nucleic acid is added, and at least one target nucleic acid is specifically and accurately measured.
[0002]
In addition, a target nucleic acid characterized by separating and recovering only a nucleic acid fraction containing a target nucleic acid base sequence after cleaving all nucleic acids containing the target nucleic acid with at least one restriction enzyme that does not cut the target nucleic acid base sequence region This is a separation / recovery concentration method.
Further, an artificially synthesized gene characterized in that gene amplification is performed using an artificially synthesized single-stranded oligonucleotide nucleic acid having an arbitrary sequence of 50 bp or more as a template to obtain a double-stranded DNA having an arbitrary sequence. It is an acquisition method.
[0003]
[Prior art]
Many methods for measuring a corresponding nucleic acid using a nucleic acid probe are known. For example,
(1) A method using a probe using a FRET (fluorescence energy transfer) phenomenon (for example, see Non-Patent Documents 1 to 3 and Patent Documents 1 and 2),
(2) Concentration of corresponding nucleic acid before amplification by a method using a probe utilizing the characteristic that fluorescent dye interacts with a specific nucleobase to reduce the amount of fluorescence emission, real-time monitoring quantitative PCR method Alternatively, a method for measuring the copy number (for example, see Non-Patent Document 4),
Many examples can be given.
[0004]
In any method, a probe labeled with a fluorescent dye is used in a measurement system in which one type of corresponding nucleic acid is present (meaning that there are a plurality of nucleic acid species in the measurement system but one corresponding nucleic acid is present). Or two types {in this case, a donor probe (sometimes simply referred to as a luminescent probe or a nucleic acid probe) and an acceptor probe (sometimes simply referred to as a quencher)} to add a hybridization reaction and / or nucleic acid An amplification reaction is performed and the fluorescence intensity of the measurement system is measured. In particular, the method using the nucleic acid amplification reaction is a method of measuring the corresponding nucleic acid before amplification by amplifying the corresponding nucleic acid by PCR in the presence of a nucleic acid probe and monitoring the PCR product in real time. It is known as a dynamic PCR method.
[0005]
In many cases, a complex sample contains an inhibitor of hybridization reaction or nucleic acid amplification reaction. In particular, in the real-time monitoring quantitative PCR method, a calibration curve is created based on the assumption that the inhibitor is not included. Therefore, in such a case, the target nucleic acid in the complex sample or nucleic acid amplification reaction before the reaction is complicated. The target nucleic acid cannot be measured accurately. In the real-time monitoring quantitative PCR method, since the corresponding nucleic acid before amplification is quantified using an exponential calibration curve, the quantification value has a large fluctuation.
[0006]
The total number of gene types present in a specific sample is enormous, and the amount is often biased to one side. In such a sample, there are often few target genes of interest. Since there is a limit to the amount of genes that can be added to a measurement system for detecting / measuring target genes (PCR methods, real-time quantitative PCR methods, etc. can be mentioned as suitable examples), up to the limit amount Even if such a gene is added, there may be a case where there are only a few target genes in the added gene or there is an undetectable amount. This often happens when detecting and measuring a target gene in a sample having the above characteristics. In this case, it becomes impossible or difficult to detect and measure the target gene accurately and specifically. For this reason, it is preferable to isolate and collect the target gene. Further, it is particularly desirable to concentrate the separated and recovered material. However, there is currently no such appropriate means.
[0007]
When producing an artificial gene having an arbitrary sequence, a single-stranded oligonucleotide (DNA) having an arbitrary sequence and an arbitrary length is synthesized using a commercially available nucleic acid synthesizer. There are many. However, since the efficiency of synthesizing one base is not 100%, the longer the base length of the oligonucleotide, the more the synthesis stops and the proportion of oligonucleotides having sequences other than the target increases. For this reason, it is generally said that the limit of synthesis of oligonucleotide is up to about 100 bases (hereinafter referred to as bp). On the other hand, when the length is about 50 bp or more, the proportion of oligonucleotides having sequences other than the target increases, and a complicated separation operation is required. Even when this operation is performed, when synthesizing a long oligo DNA of 50 bp or more, the acquisition ratio of the target oligonucleotide is often lowered as compared with the case of a short oligo DNA. For this reason, there has been a demand for a method for easily obtaining an artificial gene having an arbitrary sequence of 50 bases or more.
The present invention aims to solve the above problems.
[0008]
[Patent Document 1]
JP-A-10-262700
[Patent Document 2]
Japanese Patent Laid-Open No. 10-84983
[Non-Patent Document 1]
Morrison et al., Anal. Biochem., Vol. 183, 231-244, 1989
[Non-Patent Document 2]
Mergney et al., Nucleic acid Res., Vol. 22, 920-928, 1994
[Non-Patent Document 3]
Tyagi et al., Nature Biotech., Vol. 14, 303-308, 1996
[Non-Patent Document 4]
KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34
[0009]
[Problems to be solved by the invention]
The subject of the present invention is that, for the above purpose, when at least one kind of target nucleic acid is present in a measurement system including a complex sample, the target nucleic acid can be specifically measured by a simple method, In addition, a novel method that enables simple, accurate measurement in a short amount of time, a novel nucleic acid amplification method, a method for determining the concentration or copy number of a target nucleic acid prior to nucleic acid amplification using the method, and analyzing data obtained by these methods It is to provide methods and methods for applying these methods to various methods. Furthermore, devices such as DNA chips used in the method, measurement reagent kits, computer-readable recording media recorded with procedures for causing a computer to execute the process of the data analysis method, and target nucleic acids are measured by the method of the present invention And a measuring device incorporating a computer-readable recording medium, a method for separating / recovering and concentrating a target nucleic acid, a method for synthesizing an artificial gene having an arbitrary sequence, and the like.
[0010]
[Means for Solving the Problems]
As a result of intensive studies using the method of KURATA et al in 2) above (KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34), the base sequence of the target nucleic acid A nucleic acid (hereinafter referred to as an internal standard nucleic acid) in which a part of the target nucleic acid is mutated is added to a known concentration at a known concentration, and further, a target nucleic acid probe (hereinafter referred to as a target nucleic acid probe) that specifically hybridizes to the target nucleic acid. ) And a target nucleic acid probe that specifically hybridizes to the internal standard nucleic acid (hereinafter referred to as an internal standard nucleic acid probe) is added to the measurement system to perform a hybridization reaction and / or a nucleic acid amplification reaction. Simultaneously measure nucleic acids, or after cleaving all nucleic acids containing the target nucleic acid with at least one restriction enzyme that does not cleave the target nucleic acid base sequence region, separate only the nucleic acid fraction containing the target nucleic acid base sequence It was found that the above-mentioned problem can be solved by collecting the DNA and performing gene amplification using an artificially synthesized single-stranded oligonucleic acid of 50 bp or more having an arbitrary sequence as a template. The present invention has been completed based on such findings.
[0011]
  That is, the present invention
[Claim 1] (i) Target nucleic acid,
(Ii) having a base sequence obtained by substituting a part of the base sequence of the target nucleic acid with another base;
A known amount of an internal standard nucleic acid that can be amplified simultaneously with the target nucleic acid using the same primer,
(Iii) a target nucleic acid probe comprising an oligonucleotide having a base sequence complementary to the target nucleic acid and labeled with at least one fluorescent dyeThe Tm value of the complex of the target nucleic acid probe and the target nucleic acid has a difference of 6 ° C. or more compared with the Tm value of the complex of the target nucleic acid probe and the internal standard nucleic acid.,as well as
(Iv) What is a fluorescent dye that is composed of an oligonucleotide having a base sequence complementary to an internal standard nucleic acid and has a base sequence that does not hybridize with a target nucleic acid and that labels the target nucleic acid probe?Fluorescent colorInternal standard nucleic acid probe labeled with at least one different fluorescent dyeThe Tm value of the complex of the internal standard nucleic acid probe and the target nucleic acid has a difference of 6 ° C. or more compared to the Tm value of the complex of the internal standard nucleic acid probe and the internal standard nucleic acid. thing,
Derived from the fluorescent dyes labeled on the target nucleic acid probe and the internal standard nucleic acid probe of the reaction system.Fluorescence intensityAnd measuring the target nucleic acid and / or the target nucleic acid before the nucleic acid amplification reaction from the obtained measured value and the amount of the internal standard nucleic acid added.
[0012]
[Claim 2] (i) Target nucleic acid,
(Ii) having a base sequence obtained by substituting a part of the base sequence of the target nucleic acid with another base;
A known amount of an internal standard nucleic acid that can be amplified simultaneously with the target nucleic acid using the same primer,
(Iii) a target nucleic acid probe comprising an oligonucleotide having a base sequence complementary to the target nucleic acid and labeled with at least one fluorescent dyeThe Tm value of the complex of the target nucleic acid probe and the target nucleic acid has a difference of 6 ° C. or more compared with the Tm value of the complex of the target nucleic acid probe and the internal standard nucleic acid.,as well as
(Iv) What is a fluorescent dye that is composed of an oligonucleotide having a base sequence complementary to an internal standard nucleic acid and has a base sequence that does not hybridize with a target nucleic acid and that labels the target nucleic acid probe?Fluorescent colorInternal standard nucleic acid probe labeled with at least one different fluorescent dyeThe Tm value of the complex of the internal standard nucleic acid probe and the target nucleic acid has a difference of 6 ° C. or more compared to the Tm value of the complex of the internal standard nucleic acid probe and the internal standard nucleic acid. thing,
In the reaction system comprising: a target nucleic acid and a target nucleic acid probe, and an internal standard nucleic acid and an internal standard probe are hybridized, and before and after the reaction, the target nucleic acid probe and the internal standard nucleic acid probe are labeled with each fluorescent dye. Come fromFluorescence intensityAnd measuring the target nucleic acid from the obtained measured value and the added amount of the internal standard nucleic acid.
[0013]
[Claim 3]The target nucleic acid probe is labeled with a donor dye and an acceptor dye, and the internal standard nucleic acid probe is labeled with a donor dye that is different from the acceptor dye and a donor fluorescent dye that labels the target nucleic acid probe. Item 3. The method according to Item 1 or 2.
[0014]
[Claim 4]The target nucleic acid probe is labeled with a donor dye and a quencher dye, and the internal standard nucleic acid probe is labeled with a quencher dye and a donor dye that is different from the donor fluorescent dye that labels the target nucleic acid probe. The method according to claim 1 or 2.
[0015]
[Claim 5]The method according to any one of claims 1 to 4, wherein the fluorescent dye labeling sites of the target nucleic acid probe and the internal standard nucleic acid probe are both terminal portions of the probe.
[0018]
[Claims6The method according to claim 1, further comprising the step of decomposing the target nucleic acid probe and the internal standard nucleic acid probe by a polymerase in the nucleic acid amplification reaction.
[0019]
[Claims7The method according to claim 1, wherein the target nucleic acid probe and the internal standard nucleic acid probe are used as primers for the nucleic acid amplification reaction.
[0020]
[Claims8The method according to claim 1, wherein the method for amplifying a nucleic acid is any one of a PCR method, an ICAN method, a LAMP method, a NASBA method, an RCA method, a TAMA method, and an LCR method.
[0021]
[Claims9The PCR method is a quantitative PCR method or a real-time quantitative PCR method.8The method described in 1.
[Claim 10] The method according to claim 2, wherein the temperature of the hybridization reaction is within a range of Tm value ± 10 ° C of the complex of the target nucleic acid and the target nucleic acid probe.
[0041]
That is, the present invention
1) Specific to an internal standard nucleic acid corresponding to a target nucleic acid, a nucleic acid probe that specifically hybridizes to the target nucleic acid (hereinafter simply referred to as a target nucleic acid probe), and an internal standard nucleic acid to a measurement system containing at least one target nucleic acid A method of measuring a target nucleic acid by adding or presenting a nucleic acid probe (hereinafter simply referred to as an internal standard nucleic acid probe) that hybridizes to the target,
[0042]
2) An internal standard nucleic acid (described later) that can be suitably used in the above-described novel method for measuring nucleic acid,
3) An internal standard nucleic acid probe (described later) that can be suitably used in the above-described novel method for measuring nucleic acid,
4) A target nucleic acid probe and / or an internal standard nucleic acid probe (described later) that can be suitably used in the above-described novel method for measuring a nucleic acid,
5) A novel target nucleic acid probe and / or internal standard nucleic acid probe (described later) that can be suitably used in the novel nucleic acid measurement method of the present invention is used as a simple nucleic acid probe or a primer for nucleic acid amplification in a nucleic acid amplification method. A novel nucleic acid amplification method and a novel method for measuring the concentration or copy number of the target nucleic acid prior to nucleic acid amplification of the target nucleic acid by the method (preferred specific methods are described later),
6) A method for analyzing data obtained by the method of the present invention (a preferred specific method is described later),
[0043]
7) Reaction liquids or measurement kits that can be used in the method of the present invention and that can carry out the method of the present invention, comprising an internal standard nucleic acid, a target nucleic acid probe and / or an internal standard nucleic acid probe,
8) A plurality of target nucleic acid probes and / or internal standard nucleic acid probes that can be used in the method of the present invention are bound to the surface of the solid support, and the target nucleic acid probe and the internal standard nucleic acid probe are connected to the internal standard nucleic acid probe. And measuring the change or amount of change in the fluorescent character of the fluorescent dye labeled on the target nucleic acid probe and / or the internal standard nucleic acid probe, and devices capable of carrying out the method of the present invention, and A reaction solution or a measurement kit comprising an internal standard nucleic acid and capable of carrying out the method of the present invention using the devices;
[0044]
9) a measuring device for carrying out the method of the invention,
10) A computer-readable recording medium in which a procedure for causing a computer to execute the process of the data analysis method is recorded as a program;
11) A target characterized by separating and recovering only a nucleic acid fraction containing a target nucleic acid base sequence after cleaving all nucleic acids containing the target nucleic acid with at least one restriction enzyme that does not cleave the target nucleic acid base sequence region Nucleic acid separation / recovery concentration method,
12) An artificially synthesized gene characterized by performing gene amplification using an artificially synthesized single-stranded oligonucleotide nucleic acid of 50 bp or more having an arbitrary sequence as a template to obtain a double-stranded DNA having an arbitrary sequence Provide an acquisition method.
[0045]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in more detail with reference to preferred embodiments.
The present invention is a nucleic acid probe labeled with at least one fluorescent dye (simply referred to as a nucleic acid probe), and corresponding nucleic acid {simply hybridizable nucleic acid (not necessarily all bases are complementary) All bases do not have to be hydrogen bonded). }, In the method of measuring the corresponding nucleic acid using at least one kind of nucleic acid probe in which the fluorescent character of the labeled fluorescent dye changes (simply a nucleic acid measuring method using a nucleic acid probe), at least one kind In the measurement system containing the target nucleic acid, at least one type of internal standard nucleic acid addressed to a known concentration corresponding to the target nucleic acid (described in detail later), at least one type of target nucleic acid probe labeled with a fluorescent dye and / or internal A standard nucleic acid probe is added, a hybridization reaction and / or a nucleic acid amplification reaction is performed, and each of the hybridization reaction between the target nucleic acid probe and the target nucleic acid and / or the hybridization reaction between the internal standard nucleic acid probe and the internal standard nucleic acid is performed. Fluorescent dye labeled on the nucleic acid probe or fluorescent key of the measurement system Measure or change the amount of lactic acid before and after hybridization at at least one wavelength, and from the measured value and the concentration of the internal standard nucleic acid, the concentration of the target nucleic acid and / or the concentration or copy of the target nucleic acid before the nucleic acid amplification reaction This is a new concept of measuring the number specifically.
[0046]
Further, this method is used as a principle of the present invention to solve various problems when measuring at least one target nucleic acid, preferably in a homogeneous system, using various nucleic acid probes.
Then, all nucleic acids including the target nucleic acid are cleaved with at least one restriction enzyme that does not cleave the target nucleobase sequence region, and only the nucleic acid fraction containing the target nucleobase sequence is separated, recovered, and / or further concentrated. It is a target nucleic acid concentration method.
Furthermore, a method for preparing an artificially synthesized gene that obtains double-stranded DNA having an arbitrary sequence by artificially synthesizing a single-stranded oligonucleic acid having an arbitrary sequence of 50 bp or more and performing gene amplification using this as a template is there.
[0047]
Unless otherwise specified, terms used in the present invention have the same meaning as terms generally used in biology, molecular biology, genetics or genetic engineering, microbiology or microbial engineering, etc. is there.
In the present invention, the fluorescence character refers to fluorescence properties such as fluorescence intensity, fluorescence lifetime, fluorescence polarization, fluorescence anisotropy, etc. (hereinafter abbreviated as fluorescence intensity for simplicity).
A complex of a probe and a nucleic acid obtained by hybridization of a nucleic acid probe and a corresponding nucleic acid is called a hybrid (or hybrid) complex, or simply a nucleic acid / probe complex or a probe / nucleic acid complex.
[0048]
In addition, the measurement of nucleic acid or the concentration of nucleic acid means not only the quantitative determination of the concentration of the target nucleic acid, but also quantitative detection or simple detection.
The “method of measuring a target nucleic acid using at least one target nucleic acid probe which is a target nucleic acid probe and changes the fluorescence intensity of a labeled fluorescent dye by hybridizing to a corresponding nucleic acid” in the present invention is Simply refers to a method of measuring nucleic acid using a nucleic acid probe. It may be known or unknown. For example, the presently known method described below and the method of the present invention can be mentioned. The “corresponding nucleic acid” is as described above.
[0049]
In the present invention, the target nucleic acid refers to a nucleic acid or gene intended for nucleic acid measurement. Regardless of purification. Also, it does not matter whether the concentration is large or small. Various nucleic acids may be mixed. For example, a complex microbial system (a mixed system of RNA or genetic DNA of a plurality of microorganisms, for example, nucleic acids and genes in soil) or a symbiotic microbial system (a plurality of animal and plant and / or RNAs of a plurality of microorganisms or It is a specific nucleic acid intended for measurement in a genetic DNA mixed system). Specific examples of the nucleic acid include DNA, RNA, PNA, oligodeoxyribonucleotides, oligoribonucleotides and the like, and chemically modified nucleic acids of the nucleic acids. 2'- as a chemically modified nucleic acidO-A methyl (Me) RNA etc. can be illustrated.
[0050]
In the present invention, an internal target nucleic acid is a nucleic acid corresponding to a target nucleic acid, and preferably has at least one of the following characteristics:
1) The internal standard nucleic acid has a base sequence that can be distinguished from the target nucleic acid from the change or amount of change in the fluorescence intensity of the fluorescent dye labeled on the probe, which is generated when the nucleic acid is hybridized with the internal standard nucleic acid probe. This example is illustrated in FIGS. 2 and 3 of Example 1. That is, based on the change in the fluorescence intensity or the difference in the amount of the fluorescent dye labeled on the internal standard nucleic acid and the internal standard nucleic acid probe, and the probe nucleic acid complex of the target nucleic acid and the target nucleic acid probe or each probe of the reaction system, Differences in base sequences between target nucleic acid and internal target nucleic acid can be identified.
2) The internal standard nucleic acid hybridizes only with the internal standard nucleic acid probe that specifically hybridizes with the internal standard nucleic acid and hybridizes with the target nucleic acid probe under certain conditions (how to obtain suitable conditions will be described later). It has a non-base sequence.
3) The base sequence of the internal standard nucleic acid is partially different from that of the target nucleic acid. The partial sequence is 1 to 30, preferably 1 to 10, particularly preferably 1 to 6 in terms of the number of bases. And it is a continuous or non-continuous base sequence.
4) The base length of the internal standard nucleic acid may be different from that of the target nucleic acid.
5) The internal standard nucleic acid can be amplified simultaneously with the target nucleic acid using the same primer.
[0051]
6) The internal standard nucleic acid is, for example, a base sequence in which a part of the base sequence of the target nucleic acid is substituted or deleted with another base, and is called a mutant nucleic acid or a kind of polymorphic nucleic acid. It is what is said. A base sequence that brings about the following effects is preferable: The hybridization effect between the target nucleic acid and the target nucleic acid probe is similar or close to the hybridization effect between the internal standard nucleic acid and the internal standard nucleic acid probe (totally Need not be the same).
Similarly, the inhibition site of the substance that inhibits nucleic acid amplification of the target nucleic acid or the inhibition effect thereof is the same as or similar to that of the substance that inhibits nucleic acid amplification of the internal standard nucleic acid (need to be exactly the same) No.) It is preferred to have an inhibition site or a base sequence that brings about its inhibition effect.
The above-mentioned internal standard nucleic acid can be prepared or synthesized by the methods described in Examples 1 and 7 and nucleic acid probes described later.
[0052]
In the present invention, the “measurement system in which at least one or more target nucleic acids are present” means that one or more kinds of target nucleic acids are present in the measurement system.
When a single type of target nucleic acid is present in the measurement system, the target nucleic acid probe and / or the internal standard nucleic acid probe present in the measurement system may be singular or plural. The term “may be plural” means that one type of target nucleic acid has a plurality of sites having different base sequences, and a plurality of types of target nucleic acid probes that hybridize to the site may be used. The same can be said for the internal standard nucleic acid.
[0053]
In the present invention, when a plurality of types of target nucleic acids are present in the measurement system and / or reaction system, a plurality of types of target nucleic acid probes and / or internal standard nucleic acids and / or internal standard nucleic acid probes are present. . The target nucleic acid and the target nucleic acid probe are at least the same number in the number of types. The same applies to the internal standard nucleic acid and the internal standard nucleic acid probe. This “at least the same number” means that a base sequence site where a plurality of nucleic acid probes hybridize to one type of target nucleic acid is set, and a plurality of types of nucleic acid probes exist in the measurement system for one type of target nucleic acid. It means that it may be allowed to.
[0054]
Further, the “plural types of target nucleic acid probes, plural internal nucleic acid probes and / or internal standard nucleic acid probes” in the case where the single type and plural types of target nucleic acids are present in the measurement system. The “species internal standard nucleic acid probe” has the following meaning. That is, (a) the nucleic acid probe of the present invention is a plurality of different types of probes. For example, probes having the same form or structure of probes but different in the dyes labeled with each other can be cited as examples. (B) A combination of nucleic acid probes having different probe forms or structures, but a plurality of types of nucleic acid probes having different labeling dyes. That is, it means that a combination of nucleic acid probes having different forms described later may be used.
“Measuring the change or amount of change in fluorescence intensity of a nucleic acid probe before and after hybridization” means, for example, the difference in measured value of fluorescence intensity of a nucleic acid probe before and after hybridization, and the time of the hybridization reaction system. The rate of change in fluorescence intensity as a function. In the nucleic acid amplification reaction, it means the change or rate of change of fluorescence intensity with respect to the reaction cycle.
[0055]
The invention of the present application comprises the first to eighth inventions.
The first invention relates to a method for measuring a nucleic acid without performing a nucleic acid amplification reaction.
That is, the first invention is as described above, but in the method of measuring nucleic acid using a nucleic acid probe (whether known or not known), a measurement system or reaction system containing at least one or more target nucleic acids ( Hereinafter, it is simply abbreviated as a measurement system.) Consisting of at least one kind of internal standard nucleic acid corresponding to the target nucleic acid and one kind of oligonucleotide labeled with at least one fluorescent dye specific to the target nucleic acid. At least one kind of internal standard nucleic acid probe consisting of one kind of oligonucleotide labeled with at least one kind of fluorescent dye specific to the target nucleic acid probe or the internal standard nucleic acid, or the target nucleic acid probe and the internal standard nucleic acid probe In a measurement system comprising at least two types in total, a hybridization reaction is performed to Change or amount of fluorescence intensity of the fluorescent dye modified to the target nucleic acid probe generated by hybridization of the probe and target nucleic acid, fluorescence modified to the internal standard nucleic acid probe generated by hybridization of the internal standard nucleic acid probe and the internal standard nucleic acid The change or amount of the fluorescence intensity of the dye is measured at the same number of wavelengths as the number of nucleic acid probe species (the sum of the number of target nucleic acid probe species and the number of internal standard nucleic acid probe species). This is a novel method for measuring a nucleic acid for measuring a target nucleic acid from the measured value and the amount of an internal standard nucleic acid.
[0056]
And in this invention, the target nucleic acid probe and / or internal standard nucleic acid probe which are used suitably for the said method take the following forms.
1) A target nucleic acid probe and / or an internal standard nucleic acid probe are each a single-stranded oligonucleotide that hybridizes to a corresponding nucleic acid, and a kind of donor dye (including a reporter dye) and / or a kind of acceptor dye. Changes in the fluorescence character of the hybridization reaction system when at least one nucleic acid probe labeled (including a quencher dye or quencher substance) is hybridized to the corresponding nucleic acid. Alternatively, the donor dye and the acceptor dye are labeled on the oligonucleotide so that the amount of change increases.
[0057]
2) The target nucleic acid probe and / or internal standard nucleic acid probe of the above 1) has one of the following forms:
(1) In the form of a kind of oligonucleotide labeled with a donor dye and an acceptor dye, by hybridizing with the corresponding nucleic acid, the change or amount of change in the fluorescence character of the donor dye becomes positive before and after hybridization,
(2) The change or amount of change in the fluorescence character of the donor dye and acceptor dye is negative before and after hybridization by hybridizing with the corresponding nucleic acid in the form of a kind of oligonucleotide labeled with the donor dye and acceptor dye. What
(3) two kinds of one kind of donor probe consisting of one kind of oligonucleotide labeled with one kind of donor dye and one kind of acceptor probe consisting of one kind of oligonucleotide labeled with one kind of acceptor dye The probe is in a paired form, and the donor probe and / or the acceptor probe hybridize with the corresponding nucleic acid, so that the change or change amount of the fluorescence character of the donor dye and the acceptor dye becomes negative or positive before and after hybridization. What will be.
[0058]
3) The target nucleic acid probe and / or internal standard nucleic acid of 1) above is hybridized with the corresponding nucleic acid in the form of a kind of oligonucleotide labeled with a donor dye and an acceptor dye, so that the donor dye is before and after hybridization. And the fluorescent character of the acceptor dye changes or the amount of change is negative, and is labeled with a donor dye or an acceptor dye at its end, and the nucleic acid probe is hybridized to the corresponding nucleic acid at the end The base sequence of the probe is designed so that at least one base of G (guanine) is present in the base sequence of the corresponding nucleic acid one to three bases away from the terminal base of the corresponding nucleic acid hybridized to the probe. .
[0059]
4) The target nucleic acid probe and / or internal standard nucleic acid of 1) above is hybridized with a corresponding nucleic acid in the form of a kind of oligonucleotide labeled with a donor dye and an acceptor dye, so that the donor dye is before and after hybridization. And the fluorescent character of the acceptor dye or the amount of change is negative, and when hybridizing to the corresponding nucleic acid, at least one G of probe-nucleic acid hybrid base pairs in the donor dye or acceptor dye labeling portion The base sequence of the probe is designed so as to form a pair of (guanine) and C (cytosine).
[0060]
5) The target nucleic acid probe and / or the internal standard nucleic acid probe is composed of a single-stranded oligonucleotide labeled with at least one fluorescent dye, and the probe is designed to have at least one of the following characteristics: Has been:
(1) A function can be exhibited with a kind of nucleic acid probe in the reaction system or measurement system.
(2) When hybridized with a target nucleic acid and / or an internal standard nucleic acid, the fluorescent dye increases or changes the amount of the fluorescent character minus in the absence of the quencher dye and / or quencher probe. ,
(3) The probe is labeled at least with a fluorescent dye at its end.
[0061]
(4) When the nucleic acid probe is hybridized to the target nucleic acid, the base of the target nucleic acid is separated from the labeled base of the target nucleic acid hybridized to the probe by 1 to 3 bases (however, the labeled base is counted as 1). There is at least one base of G (guanine) in the sequence,
(5) When the nucleic acid probe hybridizes to the target nucleic acid at the terminal portion, a plurality of probe-nucleic acid hybrid base pairs form at least one G (guanine) and C (cytosine) pair at the terminal portion.
[0062]
6) The target nucleic acid probe and / or internal standard nucleic acid probe of the above 5) is phosphoric acid in which the 3′-carbon hydroxyl group of the 3′-terminal ribose or deoxyribose, or the 3′- or 2′-carbon hydroxyl group of the 3′-terminal ribose is phosphorus. It is oxidized.
7) The target nucleic acid probe and / or internal standard nucleic acid probe of 5) above is labeled with the fluorescent dye at a portion other than the OH group at the 3 ′ end, and the nucleic acid probe is hybridized to the corresponding nucleic acid. In some cases, a target nucleic acid probe and / or an internal standard nucleic acid probe in which a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair in the modified portion is used.
[0063]
The target nucleic acid probe and the internal standard nucleic acid are different so that the fluorescent color of the fluorescent dye labeling the target nucleic acid probe and the fluorescent color of the fluorescent dye labeling the internal standard nucleic acid probe are different from each other. It is preferred to design the probe. Specifically, it is preferable that the type of fluorescent dye labeling the target nucleic acid probe is different from the fluorescent dye labeling the internal standard nucleic acid probe.
[0064]
Specific examples of the nucleic acid measurement method using the above-described nucleic acid probe are shown below.
I. Examples of known methods
The currently known methods refer to all currently known methods, and are exemplified as follows. Note that the present invention is not limited by this illustration.
A) FRET When using the phenomenon:
(1) A method represented by the method of Morrison et al. (Morrison et al., Anal. Biochem., 183: 231-244, 1989)
In this method, double-stranded DNA is used as a corresponding nucleic acid, and two types of nucleic acid probes are used. One of the two types hybridizes to the leading strand and the other hybridizes to the coding strand. Further, when one of the probes is labeled with one fluorescent dye at the 5 'end, the other is labeled with one fluorescent dye at the 3' end. Further, one dye of the probe is a quencher donor dye (also called a reporter dye), and the other dye is an acceptor (also called a quencher dye). ) Dye. The nucleotide sequences of the two probes are designed so that they can hybridize with each other. When two types of probes form a hybrid complex, the FRET phenomenon acts and the fluorescence intensity of the measurement system is suppressed to a low level.
[0065]
In the measurement system, two types of probes forming a hybrid complex and a corresponding nucleic acid forming a duplex are present. Then, the probe and the corresponding nucleic acid dissociate its own hybrid complex, and the probe and the corresponding nucleic acid form a hybrid complex competitively. When the probe and the corresponding nucleic acid form a hybrid complex, the FRET phenomenon between the dyes is eliminated, and the change rate of fluorescence intensity change value or time as a function of the measurement system increases. Since the increase in the fluorescence intensity change value and the rate of change in the measurement system is proportional to the concentration of the corresponding nucleic acid, the concentration of the corresponding nucleic acid can be measured.
[0066]
In this case, first add two types of probes to the measurement system, measure the fluorescence intensity, and then add the sample containing the corresponding nucleic acid to measure the fluorescence change value or rate of change before and after. In some cases, two types of probes are added to a measurement system containing a nucleic acid, and the rate of change in fluorescence intensity as a function of time is measured. In this way, the concentration of the corresponding nucleic acid can be measured. This is because the change value or change rate of the fluorescence intensity is proportional to the concentration of the corresponding nucleic acid.
[0067]
(2) A method represented by the method of Mergney et al. (Mergney et al., Nucleic acid Res., 22: 920-928, 1994)
Two nucleic acid probes are hybridized to a single-stranded corresponding nucleic acid. When one of the two probes is labeled with one donor dye, the other is labeled with one acceptor dye. Further, when one of the probes is labeled with a fluorescent dye at the 5 'end, the other is labeled with the 3' end. The donor dye can act on the acceptor dye to increase the fluorescence emission at a specific wavelength of the acceptor dye. The donor dye is also referred to as a reporter dye, and when it acts on the acceptor dye, it weakens its own fluorescence. The acceptor dye is also referred to as a quencher dye. When two types of probes are hybridized to corresponding nucleic acids, the dye-labeled end site of one probe and the dye-labeled end site of the other probe are designed to face each other. The base sequences are designed so that the distance between the dye-labeled bases of both probes is 1 to 9 bases apart and both probes hybridize to the corresponding nucleic acid.
The actual measurement procedure is the same as the Morrison method.
[0068]
(3) Molecular beacon method (Tyagi et al., Nature Biotech., 14: 303-308, 1996; Schofield et al., Applied and Environ. Microbiol., 63: 1143-1147, 1997); Pin probe (sunrise probe) (Nazarenko, IA, Bhatnagar, SK and Hohman, RJ (1997) A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res., 25, 2516-2521); Scorpion probe (Theaker, J., Guy, SP, Brown, T. and Little, S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nature Biotechnol., 17, 804-807.)
[0069]
The above-mentioned probe is generally labeled with one fluorescent dye at the end and one quencher substance that does not emit fluorescence in other regions. When not hybridized to the corresponding nucleic acid, a stem / loop structure is formed from the homology of the base sequence in the probe molecule. By the formation of the structure, the fluorescent dye and the quencher substance are arranged at positions close to each other. This arrangement causes the FRET phenomenon and suppresses the fluorescence emission of the donor dye. However, when the probe hybridizes with the corresponding nucleic acid, the three-dimensional structure of the probe changes and the stem-loop structure is broken. This eliminates the FRET phenomenon and increases the fluorescence emission of the fluorescent dye. The concentration of the corresponding nucleic acid is proportional to the increase in the fluorescence intensity of the fluorescent dye in the measurement system. The actual measurement procedure is the same as the Morrison method.
[0070]
(4) Method represented by the method of Livak et al. (US patent No. 5,538,848)
A probe in which one quencher dye and one reporter dye are labeled at different positions at the end of a single-stranded oligonucleotide. A stem-loop structure is not formed between the base chain where the fluorescent substance is labeled and where the quencher substance is labeled. When the probe is not hybridized to the corresponding nucleic acid, the reporter dye is affected by the quencher dye to suppress fluorescence, but when hybridized to the corresponding nucleic acid, the suppression is released and the reporter dye is released. Designed to increase fluorescent color development.
The actual measurement procedure is almost the same as that of Morrison et al.
[0071]
B) A method using a probe utilizing the characteristic that a fluorescent dye interacts with a specific nucleobase to reduce the amount of fluorescence emission
(1) A method represented by the method of KURATA et al. (KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34; EP 1 046 717 A9; JP 2001-286300 (P2001-286300A)) (T.Horn, et al., Nucleic acid Research, 1997,25,4842-4849,1997; US Patent Application Publication No.US2001 / 0009760A1, Pub.Date: Jul.26,2001; US Patent No.6,140,054)
[0072]
A nucleic acid probe in which a single-stranded oligonucleotide is labeled with a single fluorescent dye, but when the site labeled with the fluorescent dye hybridizes with the corresponding nucleic acid, the base of the corresponding nucleic acid corresponding to the base of the labeled site or its The nucleic acid probe is designed so that at least one G is present in the vicinity, or a GC pair is present in the labeling site or the probe nucleic acid complex in the vicinity thereof. When the probe is hybridized to the corresponding nucleic acid, the intensity of the fluorescence emission of the hybrid complex is significantly reduced as compared with that before hybridization. Since the amount of decrease in the fluorescence intensity of the measurement system is proportional to the concentration of the corresponding nucleic acid, the corresponding nucleic acid can be measured.
The actual measurement procedure is almost the same as the method of Morrison et al., Except that the decrease value or decrease rate of the fluorescence intensity is measured. In this measurement method, measurement may be carried out by adding a helper probe for efficiently carrying out the hybridization reaction to the hybridization reaction system before the hybridization reaction.
[0073]
C) Other methods
1) A method represented by the method of Davis et al. (Davis et al., Nucleic acids Res., 24: 702-706, 1996)
The corresponding nucleic acid is measured using a probe in which a fluorescent dye is bound to the 3 'end of the oligonucleotide via a spacer having 18 carbon atoms. When the probe is hybridized to the corresponding nucleic acid, the fluorescence intensity is 10 times higher than when a probe in which the dye is directly bound to the 3 'end is used.
[0074]
2) The method represented by the method of HORN et al. (US Patent Application Publication No. US2001 / 0009760A1, Pub.Date:Jul.26,2001)
A probe consisting of a single-stranded oligonucleotide, labeled at one end with a BODIPY dye. It is designed so that a part of base sequences at both ends hybridize with each other. In the measurement system, when no corresponding nucleic acid is present, both ends are hybridized to form one loop. And the light emission of the fluorescent dye is suppressed. However, if the corresponding nucleic acid is present, the probe is hybridized to the corresponding nucleic acid, and thus the loop shape is broken. Since the fluorescent dye emits light, the concentration of the corresponding nucleic acid can be determined by measuring the fluorescence intensity.
[0075]
II. Example of nucleic acid probe of the present invention and method using the same
Summarized below.
1) Invention method A
i) In a method for measuring a corresponding nucleic acid using a nucleic acid probe labeled with a fluorescent dye, a nucleic acid probe that hybridizes to the corresponding nucleic acid and has a different emission fluorescent color in a measurement system in which at least one corresponding nucleic acid is present In the presence of at least the same number of corresponding nucleic acids, and a hybridization reaction and / or a nucleic acid amplification reaction are performed, and the fluorescence intensity of the nucleic acid probe before and after hybridization is varied at least at the same number of wavelength species as the number of nucleic acid probe species, or This is a novel method for measuring nucleic acid by measuring the increase in the positive change amount and measuring the concentration of the corresponding nucleic acid or the nucleic acid concentration or copy number before amplification.
[0076]
ii) A nucleic acid probe obtained by labeling a single-stranded oligonucleotide that hybridizes to a corresponding nucleic acid with a fluorescent dye (donor dye) and a quencher dye, wherein the nucleic acid probe hybridizes to the corresponding nucleic acid. In order to increase the fluorescence intensity of the hybridization reaction system, the fluorescent dye (donor dye) and quencher dye are labeled on the oligonucleotide, and the fluorescent dye (donor dye) is labeled and the quencher. The novel method for measuring a nucleic acid according to i) above, wherein a nucleic acid probe composed of an oligonucleotide that does not form a stem-loop structure between the base strands where the char dye is labeled is used.
[0077]
iii) The nucleic acid probe according to the above ii) further has at least one of the following characteristics.
(1) A fluorescent dye (donor dye) and a quencher dye are labeled at the same base in a single-stranded oligonucleotide.
(2) The base position of the single-stranded oligonucleotide labeled with the fluorescent dye (donor dye) and the quencher dye is the 3 'end or the 5' end.
(3) The distance between the base where the fluorescent dye (donor dye) is labeled and the base where the quencher dye is labeled is 1 to 20, or {(3 to 8 Arbitrary integer) + 10n} (where n is an integer including 0).
[0078]
(4) A fluorescent dye (donor dye) or quencher dye is labeled at the 5 'end or 3' end of the oligonucleotide, and the corresponding quencher dye or fluorescent dye is labeled in the chain.
(5) A fluorescent dye (donor dye) or a quencher dye is labeled on the 5 'end of the oligonucleotide, and a corresponding quencher dye or fluorescent dye is labeled on the 6th to 8th bases from the 5' end.
(6) The single-stranded oligonucleotide has the same chain length as the corresponding nucleic acid.
(7) The phosphate group at the 5 'end and / or 3' end of the nucleic acid probe is labeled with a fluorescent dye (donor dye).
[0079]
(8) A fluorescent dye (donor dye) that labels the nucleic acid probe is Texas red, EDANS (5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid), tetramethylrhodomine (tetramethylrhodomine) or It is at least one of its derivative, FITC or its derivative, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, or 6-TAMURA.
[0080]
(9) The quencher dye that labels the nucleic acid probe is Dabcyl (4- (4'-dimethylaminophenylazo) benzoic acid), Ferrocene or its derivatives, methyl viologen, N, N'-dimethyl-2,9-diazopyrenium At least one.
In the nucleic acid probe used in the above-described nucleic acid measurement method, one type of fluorescent dye (donor dye) and one type of quencher dye are labeled on each kind of nucleic acid probe, and at least one oligonucleotide is labeled at each site. It is a place.
[0081]
2) Invention method B
i) In a measurement system in which at least one or more corresponding nucleic acids are present in the nucleic acid measurement method, at least the same number of nucleic acid probes that hybridize to the corresponding nucleic acid and have different emission fluorescent colors and the number of the corresponding nucleic acid species are present. A nucleic acid characterized by measuring a change in fluorescence intensity of a nucleic acid probe or an increase in a negative change amount before and after hybridization at a wavelength of at least the same number as the number of nucleic acid probe species after hybridizing the probe with the corresponding nucleic acid. New measurement method.
[0082]
ii) Multiple types of nucleic acid probes so as to form at least one pair of a fluorescent dye pair that causes a FRET phenomenon, that is, a fluorescent dye that can be a donor dye (donor dye) and a fluorescent dye that can be an acceptor dye (acceptor dye) Changes in fluorescence intensity before and after hybridization with an acceptor dye in the absence of a quencher probe when the probe hybridizes to the corresponding nucleic acid in a single-stranded oligonucleotide labeled with a fluorescent dye of Alternatively, the novel method for measuring a nucleic acid according to i), wherein the base sequence is designed so that the amount of change increases negatively and the dye is labeled.
[0083]
(iii) The nucleic acid probe according to (2), wherein when the nucleic acid probe is hybridized to the corresponding nucleic acid, the change or change amount of the fluorescence intensity of the donor dye and the acceptor dye is negatively increased in the absence of the quencher nucleic acid probe. A novel method for measuring a nucleic acid according to i) or ii) above, wherein the nucleic acid probe according to i) is used.
[0084]
iv) The novel method for measuring a nucleic acid according to i), wherein the nucleic acid probe according to ii) or iii) further uses a nucleic acid probe having the characteristics according to any one of the following: .
(1) A dye that labels a nucleic acid probe is a fluorescent dye that can serve as a donor dye, and is at least one of BODIPY FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamine, or 6-TAMRA.
(2) The nucleic acid probe is labeled with a pair of dyes of a donor dye and an acceptor dye.
[0085]
(3) The nucleic acid probe is labeled with a donor dye or an acceptor dye at the end thereof, and when the nucleic acid probe is hybridized to the corresponding nucleic acid, 1 is determined from the base labeled with the donor dye or the acceptor dye in the probe. Or 3 bases apart (in this case, the terminal base is counted as 1 base. The same applies to the following invention), and the base sequence of the corresponding nucleic acid has at least 1 base of G (guanine). The base sequence of the lobe is designed.
[0086]
(4) When the nucleic acid probe is hybridized to the corresponding nucleic acid, the donor-dye labeling unit forms a plurality of probe-nucleic acid hybrid base pairs to form at least one G (guanine) and C (cytosine) pair. The base sequence of the probe is designed.
[0087]
(5) The donor dye labels the 5 'end portion (including the 5' end) of the nucleic acid probe.
(6) The donor dye labels the 3 'end portion (including the 3' end) of the nucleic acid probe.
(7) The 5 ′ terminal base of the nucleic acid probe is labeled with G or C, and the 5 ′ terminal is labeled with a donor dye.
(8) The 3 'terminal base of the nucleic acid probe is labeled with G or C, and the 3' terminal is labeled with a donor dye.
(9) The acceptor dye labels the 5 'end portion (including the 5' end) of the nucleic acid probe.
[0088]
(10) The acceptor dye labels the 3 ′ end portion (including the 3 ′ end) of the nucleic acid probe.
(11) The 5 'terminal base of the nucleic acid probe is labeled with G or C, and the 5' terminal is labeled with an acceptor dye.
(12) The 3 'terminal base of the nucleic acid probe is labeled with G or C, and the 3' terminal is labeled with an acceptor dye.
(13) When the base sequence of the nucleic acid probe binds to the corresponding nucleic acid, the base sequence of the corresponding nucleic acid is separated from the base labeled with the donor dye or acceptor dye in the probe by 1 to 3 bases. (Guanine) is designed to be present in at least one base.
[0089]
v) A nucleic acid probe comprising a single-stranded oligonucleotide labeled with a fluorescent dye, and when the nucleic acid probe is hybridized to a corresponding nucleic acid, the fluorescent dye is not a quencher probe or a quencher dye. When the change or amount of the fluorescence intensity is negatively increased below, and the probe is labeled with the fluorescent dye at the end, and the nucleic acid probe is hybridized to the corresponding nucleic acid at the end A nucleic acid whose base sequence is designed so that at least one base of G (guanine) is present in the base sequence of the corresponding nucleic acid 1 to 3 bases away from the terminal base of the corresponding nucleic acid hybridized to the probe The novel method for measuring a nucleic acid according to the above i) using a probe.
[0090]
vi) The novel method for measuring a nucleic acid according to i), wherein the nucleic acid probe according to v) uses a nucleic acid probe having the characteristics according to at least one of the following items.
(1) The nucleic acid probe is labeled with a fluorescent dye at the 3 'end.
(2) The nucleic acid probe is labeled with a fluorescent dye at the 5 'end.
[0091]
vii) a nucleic acid probe comprising a single-stranded oligonucleotide labeled with a fluorescent dye, and when the nucleic acid probe is hybridized to a corresponding nucleic acid, the fluorescent dye has a fluorescence intensity in the absence of a quencher probe. And the probe is labeled with the fluorescent dye at its end, and when the nucleic acid probe is hybridized to the corresponding nucleic acid, the end of the probe The above-mentioned i) uses a nucleic acid probe in which the base sequence of the probe is designed so that a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair in a part. A novel method for measuring nucleic acids.
[0092]
viii) The novel method for measuring a nucleic acid according to i), wherein the nucleic acid probe according to vii) uses a nucleic acid probe having the characteristics according to at least one of the following items.
(1) The 3 'terminal base of the nucleic acid probe is labeled with G or C, and the 3' terminal is labeled with a fluorescent dye.
(2) The 5 'terminal base of the nucleic acid probe is labeled with G or C, and the 5' terminal is labeled with a fluorescent dye.
[0093]
(3) The nucleic acid probe in which the 5 ′ terminal base of the nucleic acid probe is G or C and the 5 ′ terminal is labeled with a fluorescent dye is a 3 ′ carbon hydroxyl group of ribose or deoxyribose at the 3 ′ terminal, or 3 ′ The 2 ′ carbon hydroxyl group of the terminal ribose is phosphorylated.
(4) The phosphate group at the 5 'end and / or 3' end of the nucleic acid probe is labeled with a fluorescent dye.
[0094]
ix) a nucleic acid probe consisting of a single-stranded oligonucleotide labeled with a fluorescent dye, wherein the fluorescent dye changes in fluorescence intensity in the absence of a quencher nucleic acid probe or A nucleic acid probe whose amount of change increases negatively, and the probe is labeled with the fluorescent dye at a portion other than a phosphate group at the 5 ′ end or an OH group at the 3 ′ end, and the nucleic acid probe is The base sequence of the probe is such that when hybridized to the corresponding nucleic acid, a plurality of base pairs of the probe-nucleic acid complex forms at least one G (guanine) and C (cytosine) pair in the labeling portion. The novel method for measuring a nucleic acid according to i) above, which uses a designed nucleic acid probe.
[0095]
In the present invention, in order to apply the various known methods described above and the methods A and B of the present invention to the novel method for measuring nucleic acid of the present invention, the nucleic acid probe is used as at least one fluorescent dye (donor dye). It is necessary to use a kind of target nucleic acid probe and / or internal standard nucleic acid probe labeled with a fluorescent dye that can be used in the present invention, including an acceptor dye. The nucleic acid probe added to the measurement system is at least one kind of target nucleic acid probe and internal standard nucleic acid probe.
[0096]
In the present invention, “measuring a change in the fluorescence intensity of a nucleic acid probe before or after hybridization or an increase in a negative change amount” refers to an example of a measured value of a fluorescence intensity of a nucleic acid probe before and after hybridization. This refers to a negative difference or a negative change rate of fluorescence intensity as a function of the time of the hybridization reaction system. In PCR, it means the change or rate of change of fluorescence intensity as a function of reaction cycle. The simplest example of “increasing the fluorescence intensity or the amount of negative change” can be a case where the amount of decrease in the fluorescence intensity of the measurement system increases.
[0097]
For example, without using a nucleic acid probe such as a quencher probe in the method of Morrison et al. (Morrison et al., Anal. Biochem., 183: 231-244 (1989)), This means that a measured value of a change (for example, decrease) in fluorescence intensity after hybridization or a rate of decrease in fluorescence intensity as a function of time during the hybridization reaction is measured. In PCR, it means that the rate of change (for example, the rate of decrease) in fluorescence intensity as a function of the reaction cycle is measured. The term quencher probe or quencher nucleic acid probe means a nucleic acid probe that acts on a nucleic acid probe and suppresses the light emission of the nucleic acid probe. A nucleic acid probe such as a quencher probe in the method of Morrison et al. That means.
[0098]
1) The nucleic acid probe used in the method A of the present invention will be described in detail below.
The characteristic of this probe is that when it is not hybridized to the corresponding nucleic acid, the emission of the fluorescent dye is inhibited by the quencher dye, but when it is hybridized, the inhibition is released and the fluorescence intensity Is a probe that changes (eg, increases).
[0099]
In the present invention, the fluorescent substance is a type of fluorescent dye that is generally labeled on a nucleic acid probe and used for measurement / detection of nucleic acid. For example, fluorescein or derivatives thereof (eg, fluorescein isothiocyanate (FITC) or derivatives thereof, such as Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS, rhodamine 6G ( R6G) or its derivatives (eg, tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine, Texas red, BODIPY ) FL (BODIPY is a trade name, FL is a trade name; manufactured by Molecular Probes, USA; the same applies hereinafter), BODIPY FL / C3, BODIPY FL / C6, Bodepy ( BODIPY) 5-FAM, BODIPY TMR, or derivatives thereof (eg, BODIPY TR), BODIPY (BODI PY) R6G, BODIPY 564, BODIPY 581, 6-TAMURA, etc. Among these, FITC, EDANS, Texas Red, 6-joe, TMR, Alexa 488, Alexa 532, Bodepy (BODIPY) FL / C3, BODIPY R6G, BODIPY FL, Alexa532, BODIPY FL / C6, BODIPY TMR, 5-FAM, BODIPY 493/503, BODIPY 564, BODIPY 581, 6-TAMURA, Cy3, Cy5, Texas red, x-Rhodamine and the like can be mentioned as suitable ones.
[0100]
The quencher substance is a substance that acts on the fluorescent substance to suppress or quench the light emission. For example, Dabcyl, QSY7 (Molecular Probe), QSY33 (Molecular Probe), Ferrocene or a derivative thereof, methyl viologen, N, N'-dimethyl-2,9-diazopyrenium, and preferably Dabcyl .
By labeling the fluorescent substance and the quencher substance at a specific position of the oligonucleotide as described above, the luminescence of the fluorescent substance is quenched by the quencher substance.
[0101]
In the present invention, the nucleic acid probe of the present invention is formed, and a single-strand that does not form a stem-loop structure between the base chain where the fluorescent substance is labeled and the quencher substance is labeled. Oligonucleotides are double-stranded in the self-strand due to the complementarity of the base sequences at at least two locations between the base strand where the fluorescent substance is labeled and the location where the quencher substance is labeled. An oligonucleotide that forms a strand and does not form a stem-and-loop structure.
[0102]
When the nucleic acid probe of the present invention is hybridized to the corresponding nucleic acid, the fluorescent substance and the quencher substance are labeled on the oligonucleotide of the present invention so that the fluorescence intensity of the hybridization reaction system changes (eg, increases). To do this, you can do as follows: The distance between the base where the fluorescent substance is labeled and the base where the quencher substance is labeled is zero in terms of the number of bases, i.e., the same nucleotide of the single-stranded oligonucleotide of the fluorescent substance and the quencher substance. It is labeled at a location, or 1 to 20 in terms of the number of bases, or {(any integer from 3 to 8) + 10n} (where n is an integer including 0). Preferably, 10 is added to the same nucleotide position of a single-stranded oligonucleotide, or 3 to 8 or any number thereof. More preferably, it is the position of the same nucleotide of a single-stranded oligonucleotide, or 3-8. Thus, each substance is preferably labeled with an oligonucleotide. However, the base interval strongly depends on the base sequence of the probe, the fluorescent substance and quencher substance used for labeling, the length of the linker that binds them to the oligonucleotide, and the like. Therefore, it is difficult to completely specify the base interval, and the above-described base interval is merely a general example, and there are many exceptional cases.
[0103]
When labeling at the same nucleotide site of a single-stranded oligonucleotide, the labeling site should be labeled with one side as a base and the other side as a portion other than a base, that is, a phosphate moiety, or a ribose or deoxyribose moiety. Is preferred. In this case, it is preferable to label the 3 'end or 5' end.
[0104]
Alternatively, when the distance between the base for labeling the fluorescent substance and the quencher substance is set as described above, each substance may be labeled in the oligonucleotide chain, and one of them may be labeled at the 5 ′ end or 3 ′ end of the oligonucleotide. And other corresponding substances may be labeled in the chain. Preferably, the fluorescent substance or the quencher substance is labeled at the 5 'end or 3' end of the oligonucleotide, and the corresponding quencher substance or the fluorescent substance is labeled from the end at the above-mentioned base number interval. In this case, when labeling the 3 ′ end part or the 5 ′ end part, the base, the phosphate part, or the ribose part or deoxyribose part, preferably the phosphate part, ribose part or deoxyribose part, more preferably It is better to label the phosphate part. When labeling in the chain, it is preferable to label the base in the chain.
[0105]
2) The nucleic acid probe used in the nucleic acid measurement method i) to iii) of the present invention method B will be described in detail below.
The donor dye that can be a donor dye in the present invention includes at least a. Excited at a specific wavelength and emitting at a specific wavelength, b. The luminescence energy can be transferred to a specific dye (a dye that can be an acceptor dye), c. A GC base pair complex (GC base pair hydrogen bond) generated when a nucleic acid probe is hybridized with a corresponding nucleic acid (hereinafter sometimes referred to as a GC hydrogen bond for convenience) is in the vicinity of a donor dye. When present, it is defined as satisfying a condition such that energy can be transferred toward the base pair. In other words, any pigment that satisfies this condition may be used. In general, dyes that can serve as donor dyes in the FRET phenomenon are preferably used (Nucleic) in which the fluorescence intensity of the probe changes (eg, decreases) when a nucleic acid probe that is labeled by itself is hybridized to the corresponding nucleic acid. Acid, 29, No.6 e34, 2001).
[0106]
The dye used is specifically the same as the dye used in the present invention A. Among them, BODIPY FL, the BODIPY FL dye, BODIPY 493/503), 5- More preferred examples of FAM, BODIPY 5-FAM, Tetramethylrhodamine, 6-TAMRA, and the like include BODIPY FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamine, and 6-TAMRA. However, the present invention is not limited to these examples.
[0107]
In addition, acceptor dyes are generally dyes that can become acceptor dyes in pairs with donor dyes in the FRET phenomenon, that is, they can undergo energy transfer from donor dyes (in other words, quenching (quenching action) on donor dyes) Any pigment can be used. It depends on the type of donor dye forming the pair. For example, if BODIPY FL, the BODIPY FL dye, BODIPY 493/503, 5-FAM, BODIPY 5-FAM, Tetramethylrhodamine, 6-TAMRA, etc. are used as donor dyes, rhodamine (rhodamine) ) X, BODIPY 581/591, etc. can be used as acceptor dyes. However, the present invention is not limited to these examples.
[0108]
The structure of a preferred nucleic acid probe is labeled with a donor dye at its end, and when the nucleic acid probe hybridizes to the corresponding nucleic acid at the end, 1 to 3 from the terminal base of the corresponding nucleic acid hybridized to the probe. This means that the base sequence of the probe is designed so that there is at least one base of C (cytosine) or G (guanine) in the base sequence of the corresponding nucleic acid apart from the base.
[0109]
More preferably, when the nucleic acid probe is hybridized to the corresponding nucleic acid, a plurality of base pairs of the probe-nucleic acid hybrid form at least one G (guanine) and C (cytosine) pair at the donor dye label. The base sequence of the probe is designed.
Particularly preferably, the donor dye labeling site is a G (guanine) or C (cytosine) base, or a phosphate group of a nucleotide having the base, or an OH group of ribose.
[0110]
The labeling site of the donor dye and the acceptor dye is the acceptor dye or the donor dye when the donor dye or the acceptor dye labels the base, the phosphate part or the deoxyribose part, or the ribose part at the 5 ′ end of the nucleic acid probe. The dye is in the strand of the nucleic acid probe or at the 3 ′ end (including the 3 ′ end base). In addition, when the donor dye is labeling the base, phosphate part or deoxyribose part, or ribose part at the 3 ′ end of the nucleic acid probe, the donor dye or acceptor dye is in the nucleic acid probe chain or at the 5 ′ end. Part (including 5 ′ terminal base). When labeling the terminal portion, it may be labeled with both dyes. That is, for example, the phosphoric acid part may be labeled on one side, and the deoxyribose part or the ribose part or the base part may be labeled on the other side (for example, the distance between the bases of the labeling part of the donor dye and the acceptor dye is 0). Case (described below)). Alternatively, a spacer having a side chain may be used to bind both to a single spacer. In the present invention, it is a matter of course that all the above conditions may be satisfied even if the donor dye and the acceptor dye are both labeled in the chain.
The binding position of the fluorescent dye at each site is preferably bonded to the OH group or amino group via a spacer.
[0111]
The distance between the label part of the donor dye and the acceptor dye depends essentially on the kind of the dye pair of the donor dye and the acceptor dye, but generally 0 to 50 bases, preferably 0 to 40 bases, more preferably. Is 0 to 35 bases, particularly preferably 0 to 15 bases. If it exceeds 50 bases, the FRET phenomenon becomes unstable. At 15 to 50 bases, the fluorescence intensity of the acceptor dye changes (eg, decreases), but the fluorescence intensity of the donor dye may also change (eg, increase). That is, when the nucleic acid probe is hybridized to the corresponding nucleic acid, the three-dimensional structure of the nucleic acid probe may change. The change may eliminate the FRET phenomenon between the nucleic acid probe dyes. Depending on the magnitude of the quenching action of the acceptor dye on the donor dye (quenching action) and the quenching action of the GC hydrogen bond on the donor dye, the fluorescence intensity of the donor dye increases from before the hybridization, Or decrease. When the quenching action of the GC hydrogen bond is larger, it decreases, but when it is small, it may increase.
[0112]
Therefore, in a preferred form of the probe of the present invention, the labeling part of the donor dye and the acceptor dye has an interbase distance, and the donor dye is BODIPY FL, BODIPY 493/503, 5-FAM, Tetramethylrhodamine, or 6-TAMRA, The acceptor dye is 6-TAMRA, BODIPY 581/591, X-rhodamine, the 5 ′ terminal base is G or C, and the 5 ′ terminal is labeled with a donor dye or an acceptor dye, or a nucleic acid probe In which the 3 ′ terminal base is G or C and the 3 ′ terminal is labeled with a donor dye or an acceptor dye. A particularly preferred form is that in which the base of the corresponding nucleic acid corresponding to the dye labeling site is G. In this case, the base of the dye labeling site is not necessarily C.
[0113]
The structure of the novel nucleic acid probe for nucleic acid measurement of the present invention is as described above. In such a structure, when the energy of the excited donor dye is not hybridized to the corresponding nucleic acid, it is transferred to the acceptor dye, so that the acceptor dye emits light and has a strong fluorescence intensity. Yes. Therefore, since the emission of the donor dye is suppressed, the fluorescence intensity is kept at a low level. However, when the nucleic acid probe hybridizes to the corresponding nucleic acid, the energy of the donor dye or acceptor dye is transferred to the hydrogen bond of GC generated by the probe-nucleic acid hybrid complex or to G of the corresponding nucleic acid.
[0114]
In addition, when the nucleic acid probe of the present invention is hybridized to the corresponding nucleic acid, the fluorescence intensity of the luminescence of the nucleic acid probe is significantly reduced as compared with that before hybridization. Further, in the present invention, a plurality of acceptor dyes can be combined with one kind of donor dye, so that the number of nucleic acid probes of the combination can be obtained. And it has the said property.
[0115]
In actual nucleic acid measurement, since the decrease in the fluorescence intensity of the acceptor dye is measured, multiple types of nucleic acid probes can be used simultaneously with one excitation wavelength. That is, when many kinds of corresponding nucleic acids exist in the same measurement system, if such a nucleic acid probe is added simultaneously, many kinds of nucleic acids can be measured simultaneously with one excitation wavelength. That is, many types of nucleic acids can be measured simultaneously with a simple apparatus.
In addition, in the nucleic acid probe used in the nucleic acid measurement method according to the above i) to iii), each of the fluorescent dye and the quencher dye labeled on one nucleic acid probe is one each, and the oligonucleotide labeled sites are each There is at least one location.
[0116]
3) The nucleic acid probe used in the nucleic acid measurement method of v) to ix) of the present invention method B will be described in detail below.
It is a nucleic acid probe used in a method typified by the method of KURATA et al.
The feature of this nucleic acid probe is that it consists of a single-stranded oligonucleotide labeled with at least one fluorescent dye. When hybridized to the corresponding nucleic acid, the quencher probe or quencher dye is not present (measurement system). In the absence of the quencher probe, ie, without interaction with the quencher dye, the fluorescence intensity of the nucleic acid probe changes (eg, decreases).
In principle, this is based on the phenomenon in which the luminescence energy of the nucleic acid probe is transferred to the GC pair (hydrogen bond complex) of the probe and the corresponding nucleic acid, particularly G of the corresponding nucleic acid, and the luminescence is suppressed.
[0117]
The number of bases of the probe of the present invention is the same as that of the above invention. The base sequence of the probe is not particularly limited as long as it specifically hybridizes to the corresponding nucleic acid. Preferably, when the nucleic acid probe is labeled with a fluorescent dye and hybridizes to the corresponding nucleic acid,
(1) a base sequence in which the base sequence of the probe is designed so that at least one base of G (guanine) is present in the base sequence of the corresponding nucleic acid at a distance of 1 to 3 bases from the fluorescently labeled base;
(2) The base sequence of the probe is 1 to 3 bases away from the terminal base of the corresponding nucleic acid hybridized to the probe so that there is at least one base (G) in the base sequence of the corresponding nucleic acid. Designed base sequence,
[0118]
(3) A plurality of base pairs of the nucleic acid hybrid complex form at least one pair of G (guanine) and C (cytosine) at the terminal portion of the probe, or the base of the corresponding nucleic acid at the fluorescent dye labeling site A base sequence in which the base sequence of the probe is designed so that is G;
(4) In a probe labeled with a fluorescent dye in a portion other than the 5′-terminal phosphate group and the 3′-terminal 0H group, the base pair of the portion labeled with the fluorescent dye has at least one pair of G (guanine) and C A base sequence in which the base sequence of the probe is designed so as to form a (cytosine) pair or so that the base of the corresponding nucleic acid at the fluorescent dye labeling site is G is preferable.
[0119]
The fluorescent dye labeled on the oligonucleotide is the same as described above. Among the above descriptions, FITC, EDANS, Texas red, 6-joe, TMR, x-rhodamine, Cy3, Cy5, Alexa 488, Alexa 532, 5-FAM, BODIPY FL, BODIPY 493/503, BODIPY R6G, BODIPY 564, BODIPY 581, BODIPY FL / C3, BODIPY FL / C6, BODIPY TMR, 6-TAMURA, etc. are suitable Among them, in particular, 5-FAM, BODIPY 493/503, BODIPY FL, 6-joe, TMR, 6-TAMURA, etc. are more suitable (fluorescence intensity reduction rate is 60%) It has the above.
The method of labeling the oligonucleotide with a fluorescent dye is the same as in the above invention.
It is also possible to introduce a fluorescent dye molecule into the chain of the probe nucleic acid (ANALYTICAL BIOCHEMISTRY 225, pages 32-38 (1998)).
[0120]
Preferred probe forms are those in which the 3 ′ or 5 ′ end is labeled with a fluorescent dye, and the base at the labeled end is G or C, or the base of the corresponding nucleic acid at the fluorescent dye labeling site is It is G. When the 5 ′ end is labeled and the 3 ′ end is not labeled, the OH group at the 3′-position carbon of the ribose or deoxyribose at the 3′-end is a phosphate group, etc., and the 2′-position carbon at the 3′-end ribose The OH group may be modified with a phosphate group or the like without any limitation.
Moreover, you may modify the base in a probe chain | strand, especially C or G.
In addition, in the nucleic acid probe used in the nucleic acid measurement method described in v) to ix) above, at least one fluorescent dye is labeled on one nucleic acid probe, and at least one labeled portion is present.
[0121]
The nucleic acid probes used in the nucleic acid measurement methods A and B of the present invention (hereinafter, for the sake of simplicity, each may be referred to as a nucleic acid probe A or a nucleic acid probe B) are composed of oligodeoxyribonucleotides or oligoribonucleotides. May be. It may also be a chimeric oligonucleodite containing both of them. Those oligonucleotides may be chemically modified. Chemically modified oligonucleotides may be interposed in the chain of the chimeric oligonucleotide.
[0122]
As the modification site of the oligonucleotide subjected to the above-mentioned chemical modification, the terminal hydroxyl group or terminal phosphate group at the end of the oligonucleotide, or the nucleoside phosphate site in the chain, the carbon at the 5-position of the pyrimidine ring, and the nucleoside sugar (ribose) Or a deoxyribose) site | part can be mentioned. Preferable examples include ribose or deoxyribose sites.
[0123]
The oligonucleotide of the nucleic acid probe of the present invention can be produced by a general method for producing an oligonucleotide. It is also preferable to synthesize using a commercially available nucleic acid synthesizer (for example, ABI394 (Perkin Elmer, USA)).
[0124]
In order to label the oligonucleotide with a fluorescent dye, any desired labeling method known in the art can be used (Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63). , 1143-1147, 1997; Nucleic acids Research, 24, 4532-4535, 1996). For example, when a fluorescent dye molecule is bound to the 5 'end, first, as a spacer to the phosphate group at the 5' end, for example,-(CH₂)_nIntroduce -SH. These introducers are commercially available and may be purchased commercially (Midland Certified Reagent Company). In this case, n is 3-8, preferably 6. An oligonucleotide labeled with a fluorescent dye can be synthesized by binding a fluorescent dye having SH group reactivity or a derivative thereof to this spacer. The oligonucleotide labeled with the fluorescent dye synthesized as described above can be purified by chromatography such as reverse phase to obtain the nucleic acid probe of the present invention.
[0125]
The 3 'terminal base of the oligonucleotide can also be labeled.
In this case, as a spacer to the OH group at the 3'-position C of ribose or deoxyribose, for example,-(CH₂)_n-NH₂Is introduced. Since these introduced bodies are also commercially available in the same manner as described above, commercially available products may be purchased. In addition, by introducing a phosphate group, as a spacer to the OH group of the phosphate group, for example,-(CH₂)_nIntroduce -SH. In these cases, n is 3-8, preferably 4-7. An oligonucleotide labeled with a fluorescent dye can be synthesized by binding a fluorescent dye having reactivity with an amino group or SH group or a derivative thereof to the spacer.
[0126]
In addition, the base in the chain of the oligonucleotide can be labeled.
The base amino group or OH group may be labeled with the dye of the present invention in the same manner as at the 5 'or 3' end (ANALYTICAL BIOCHEMISTRY 225, pages 32-38 (1998)).
When introducing into an amino group, kit reagents (for example, Uni-link aminomodifier (CLONTECH, USA), Fluo Reporter Kit F-6082, F-6083, F-6084, F-10220 (any It is also convenient to use a molecular probe (Molecular Probes, USA). Then, a fluorescent dye molecule can be bound to the oligoribonucleotide according to a conventional method.
The oligonucleotide synthesized in this manner can be purified by chromatography such as reverse phase to obtain the nucleic acid probe of the present invention.
[0127]
The internal standard probe of the present invention utilizes the above-described nucleic acid probe and has the following characteristics:
1) Labeled with at least one fluorescent dye.
2) The internal standard nucleic acid probe has a sequence that hybridizes only with the internal standard nucleic acid under certain conditions and does not hybridize with the target nucleic acid.
3) The fluorescent dye labeled on the target nucleic acid probe, which is produced when the fluorescent intensity of the labeled fluorescent dye changes or changes due to hybridization with the internal standard nucleic acid when the target nucleic acid probe hybridizes with the target nucleic acid. It can be clearly distinguished from the change or amount of the fluorescence intensity.
[0128]
4) (1) When the internal standard nucleic acid probe forms a probe-nucleic acid complex with the target nucleic acid:
The base sequence of the internal standard nucleic acid indicates that the Tm value of the probe / nucleic acid complex of the internal standard nucleic acid probe and the internal standard nucleic acid is compared with the Tm value of the probe / nucleic acid complex of the internal standard nucleic acid probe and the target nucleic acid. It has a difference of not less than 10 ° C, preferably not less than 6 ° C, more preferably not less than 10 ° C.
(2) When the target nucleic acid probe forms a probe / nucleic acid complex with the internal standard nucleic acid:
The base sequence of the internal standard nucleic acid is such that the Tm value of the probe / nucleic acid complex is 3 ° C or higher, preferably 6 ° C or higher, compared to the Tm value of the probe / nucleic acid complex of the target nucleic acid probe and the target nucleic acid. Preferably, it has a difference of 6 ° C. or more.
[0129]
The target nucleic acid probe has a base sequence that specifically hybridizes to the target nucleic acid, and has a base sequence that does not specifically hybridize to the internal standard nucleic acid.
Similarly, an internal standard nucleic acid probe has a base sequence that specifically hybridizes to an internal standard nucleic acid, and has a base sequence that does not specifically hybridize to a target nucleic acid.
In the present invention, at least one of the above probes is used for one kind of target nucleic acid or internal standard nucleic acid.
The internal standard nucleic acid, internal standard nucleic acid probe, target nucleic acid, and target nucleic acid probe are as described above. The same applies to the nucleic acid measurement method using the nucleic acid amplification method described later. However, this is an example, and the present invention is not limited to these examples.
[0130]
The present invention provides a measurement system comprising at least one target nucleic acid probe and / or at least one internal standard nucleic acid probe as described above, and at least one target nucleic acid together with at least one internal standard nucleic acid at a known concentration. Add and let the hybridization reaction occur and measure the change or amount of fluorescence intensity of the fluorescent dye labeled on the target nucleic acid probe and / or internal standard nucleic acid probe of the measurement system or hybridization reaction system at at least one wavelength Then, the target nucleic acid concentration is measured from the measured value and the concentration of the internal standard nucleic acid.
In the present invention, the measurement system or the hybridization reaction system may be a solution system or a solid system.
[0131]
Hybridization reaction conditions may be performed under normal known conditions. In particular, for temperature, it is preferable to conduct an experiment of the following procedure, as shown in Example 1, to determine suitable temperature conditions:
(1) A dissociation curve between a target nucleic acid and a target nucleic acid probe or an internal standard nucleic acid probe is obtained.
(2) Obtain a dissociation curve between the internal standard nucleic acid and the internal standard nucleic acid probe or the target nucleic acid probe.
(3) From the above dissociation curve, the target nucleic acid and the target nucleic acid probe are hybridized, but the temperature at which the probe nucleic acid probe complex of the target nucleic acid and the internal standard nucleic acid probe is dissociated, and the internal standard nucleic acid and the internal The temperature at which the standard nucleic acid probe is hybridized but the internal standard nucleic acid and the target nucleic acid probe / nucleic acid probe complex are dissociated is determined. The temperature common to both is the hybridization temperature.
The buffer solution, metal ions, and the like may be under normal known conditions.
[0132]
The “change amount” is more specifically exemplified as follows. The difference in fluorescence intensity or the rate of change in fluorescence intensity in a specific measurement wavelength in a measurement system before and after hybridization between a target nucleic acid and a target nucleic acid probe or an internal standard nucleic acid and an internal standard nucleic acid probe. In this case, the measurement wavelength is at least one or more. This comes from the procedure of measuring at least one wavelength of one fluorescent dye labeling one nucleic acid probe.
The rate of change in fluorescence intensity is the amount of change in fluorescence intensity relative to the fluorescence intensity before the reaction as a function of time after the start of the hybridization reaction, the rate of change, and the fluorescence before the reaction as a function of the number of cycles in PCR. The rate of change in fluorescence intensity after the start of reaction with respect to the intensity value.
[0133]
“Change or change in fluorescence character of target nucleic acid probe caused by hybridization between target nucleic acid probe and target nucleic acid, change or change in fluorescence intensity of internal standard nucleic acid probe caused by hybridization between internal standard nucleic acid probe and internal standard nucleic acid “Measure the target nucleic acid from the amount and the added amount of the internal standard nucleic acid” means, for example, the amount or concentration of the internal standard nucleic acid at a known concentration and the internal standard nucleic acid when the internal standard nucleic acid probe is hybridized with the internal standard nucleic acid. The relationship between the change or the amount of change in the fluorescence intensity of the probe is graphed so as to be visible or a mathematical relational expression, and the change or change in the fluorescence intensity of the target nucleic acid probe is represented in a graph or relational expression. This refers to determining the amount or concentration of the target nucleic acid by insertion. Alternatively, the procedure for determining the amount or concentration of the target nucleic acid from the graph or the relational expression is recorded on a computer-readable recording medium, and the fluorescence derived from the target nucleic acid probe when the target nucleic acid and the target nucleic acid probe are hybridized. This refers to determining the amount or concentration of a target nucleic acid by inputting a change or amount of intensity into a computer.
[0134]
In particular,
1) First, in a reaction system (measurement system) that is considered not to inhibit hybridization reaction between nucleic acids and nucleic acid amplification reaction (here, reaction using a nucleic acid probe; the same applies hereinafter), a target with a known concentration. Nucleic acids and probes corresponding to the internal target nucleic acid are added to perform each reaction.
2) Measure the amount or rate of change in fluorescence intensity of the reaction system (measurement system) derived from the hybridization reaction and nucleic acid amplification reaction.
3) Graph or formulate the relationship between each nucleic acid concentration and the amount or rate of change of the fluorescence intensity.
4) A hybridization reaction and a nucleic acid amplification reaction are performed using an unknown sample containing at least one target nucleic acid, a target nucleic acid probe, internal standard nucleic acid at various known concentrations, and an internal standard nucleic acid probe. Then, the amount of change and the rate of change in the fluorescence intensity of the reaction system derived from each fluorescent dye labeled on the target nucleic acid probe and the internal standard nucleic acid probe of the reaction system are measured and calculated.
[0135]
5) Graph or formula showing the relationship between the amount of change in the fluorescence intensity of the reaction system derived from the fluorescent dye of the internal standard nucleic acid probe in 4) above, the rate of change and the concentration of the internal standard nucleic acid or the concentration of the internal standard nucleic acid before amplification. Turn into.
6) Compare and examine the graph or mathematical expression of the internal standard nucleic acid obtained in 3) above with that obtained in 5) above. When there is no difference, the graph or the mathematical formula obtained in 3) can be used as it is. If there is a difference, the coefficient of difference is calculated manually or by a computer.
7) The graph or mathematical formula for the target nucleic acid of 3) is corrected using the coefficient.
8) Apply the amount or amount of change in the fluorescence intensity for the target nucleic acid obtained in 4) above to the corrected graph or formula, and the concentration or copy of the target nucleic acid in the unknown sample, or before amplification of the target nucleic acid. Determine the density or copy number.
[0136]
The method for obtaining the concentration of the target nucleic acid has various variations and is not limited to the above example.
For example, a known amount of an internal standard nucleic acid is added to an unknown sample. A hybridization reaction is performed using the target nucleic acid probe and the internal standard nucleic acid probe. The amount of change in fluorescence intensity of the fluorescent dye labeled on each probe is measured. The relationship between the amount of change for the internal standard nucleic acid and the concentration of the internal standard nucleic acid is considered to be applicable to the target nucleic acid (Note that this can be done in a system where there is no hybridization inhibitor or polymorphism. Check). Therefore, a relational expression between the change amount of the internal standard nucleic acid and the internal standard nucleic acid concentration is obtained in advance using the unknown sample. From the relational expression, the concentration of the target nucleic acid in the unknown sample can be estimated.
[0137]
A feature of the present invention is that the fluorescence intensity is measured at at least one wavelength. For example, when one target nucleic acid is present in one measurement system, at least two types of target nucleic acid and the internal standard nucleic acid probe that hybridizes to the target nucleic acid and the target nucleic acid probe that hybridizes to the target nucleic acid are measured simultaneously. Is required. When there are two types of target nucleic acids, the measurement wavelength is at least four wavelengths.
[0138]
However, in the present invention, it is not always necessary to perform the measurement simultaneously. That is, when measuring changes or changes in the fluorescence intensity of each probe during hybridization of an internal standard nucleic acid and an internal standard nucleic acid probe, or between a target nucleic acid and a target nucleic acid probe, they are not necessarily performed at the same time, but on separate measurement systems. May be. In this case, at least one measurement wavelength may be used. Even if the measurement is performed separately, the measurement system preferably contains the target nucleic acid or the internal standard nucleic acid. Of course, the method of the present invention can be carried out even if these nucleic acids are not contained.
[0139]
In the present invention, at least one type of probe may exist in one target nucleic acid. This means that the base sequence in which a plurality of types of probes hybridize to one target nucleic acid overlaps the hybridized region. There may be more than one. In this case, the measurement wavelength increases by the type of probe.
In the present invention, the measurement conditions may be the above-mentioned known conditions, and may be changed as appropriate according to the method for measuring the target nucleic acid.
[0140]
Next, the second invention of the present invention will be described below.
According to a second aspect of the present invention, there is provided a measurement system or nucleic acid amplification reaction system containing at least one target nucleic acid, the at least one internal standard nucleic acid having a known concentration, at least one target nucleic acid probe, and / or at least one internal standard nucleic acid. Changes or changes before and after the nucleic acid amplification reaction in each cycle of the fluorescence intensity of the fluorescent dye labeled with each nucleic acid probe by amplifying the target nucleic acid and / or the internal standard nucleic acid by a nucleic acid amplification method with the addition or presence of a probe This is a method of measuring the amount in real time and measuring the concentration or copy before amplification of the target nucleic acid from the measured value and the concentration of the internal standard nucleic acid.
[0141]
The nucleic acid amplification method referred to in the present invention refers to a method for amplifying a nucleic acid in vitro.
For example, it does not matter whether it is known or not known. For example, PCR method, quantitative PCR method, LCR method (ligase chain reaction), real time monitoring quantitative polymerase chain reaction assays, TAS method, RT-PCR, RNA-primed PCR, Stretch PCR, Inverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, PCR using PNA, etc. (protein / nucleic acid / enzyme: Volume 35, No. 17, 1990, Kyoritsu Shuppan Co., Ltd .; experiment Medicine, Vol.15, No.7, 1997, Yodosha; Guide to using the PCR method, edited by Yoshio Yazaki, 1998, Chugai Medical), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) method, LAMP method , NASRA method, RCA method, TAMA method, UCAN method, etc. shall be included. Further, the term “quantitative” means not only the original quantitative measurement but also the quantitative measurement of the degree of detection as described above. It should be noted that a melting curve analysis or analysis method is also included.
[0142]
I) Known methods
As an example, a PCR method will be described.
PCR methods can be divided into the following two cases depending on how the probes are used.
A) When a nucleic acid probe is simply hybridized to an amplification product by PCR and a change in fluorescence intensity is simply used as a signal (Witwer et al., US Patent No. 6,174,670 B1; Livak et al., US Patent No. 5,538,848) ; KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34; EP 1 046 717 A9; JP 2001-286300 (P2001-286300A)); US Patent No. 6,495,326)
[0143]
In this case, there are two cases depending on how the nucleic acid probe is used.
a) Method using two nucleic acid probes
According to the method of Morrison et al. (Morrison et al., Anal. Biochem., 183: 231-244, 1989) and the method of Mergney et al. (Mergney et al., Nucleic acid Res., 22: 920-928, 1994) This is a representative method. That is, a method using a donor nucleic acid probe and an acceptor nucleic acid probe.
(A) In the method of Mergney et al., Two nucleic acid probes are hybridized to the amplified nucleic acid. Then, the fluorescence intensity of the acceptor probe of the nucleic acid probe during the annealing reaction and the fluorescence intensity of the acceptor dye released from the amplified nucleic acid after the nucleic acid probe is decomposed by the DNA polymerase during the nucleic acid extension reaction are measured. This is a method of measuring the difference between the measured values in each cycle in real time.
(B) Morrison et al.
The structure, action, and measurement are as described in the first invention and the above.
[0144]
b) When using a single nucleic acid probe
This case is also divided into the following three cases.
(A) Molecular beacon method (Tyagi et al., Nature Biotech., 14: 303-308, 1996; Schofield et al., Applied and Environ. Microbiol., 63: 1143-1147, 1997); Pin probe (Sunrise probe) (trade name: Amplifluor haipin primers: Amplifluor haipin primers; Nazarenko, IA, Bhatnagar, SKand Hohman, RJ (1997) A closed tube format for amplification and detection of DNA based on energy transfer.Nucleic Acids Res , 25,2516-2521.); Scorpion probe ((Whitcombe, D., Theaker, J., Guy, SP, Brown, T. and Little, S. (1999) Detection of PCR products using self-probing amplicons and Fluorescence. Nature Biotechnol., 17, 804-807.).
[0145]
A kind of oligonucleotide is labeled with a donor fluorescent dye and an acceptor fluorescent dye at different sites of the oligonucleotide. Based on the three-dimensional structure of the oligonucleotide, both dyes cause the FRET phenomenon. When an amplified nucleic acid is encountered, the three-dimensional structure is broken and hybridizes to the amplified nucleic acid. Due to the three-dimensional structure change, the phenomenon of FRET between the dyes is eliminated. The nucleic acid probe hybridized to the amplified nucleic acid has the same fate as described above. Then, the difference in fluorescence intensity is measured as described above.
[0146]
(B) The method represented by the method of Livak et al. (US patent No. 5,538,848)
The structure, action, and measurement are as described in the first invention and the above.
(C) A method represented by the method of KURATA et al. (KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34; EP 1 046 717 A9; JP 2001-286300 (P2001-286300A)) (T.Horn, et al., Nucleic acid Research, 1997,25,4842-4849,1997; US Patent Application Publication No.US2001 / 0009760A1, Pub.Date: Jul.26,2001; US Patent No.6,140,054) This is a representative method.
The structure, action, and measurement are as described in the first invention and the above.
[0147]
B) When using the specific nucleic acid probe as a primer for PCR
This is a method according to the method of KURATA et al. (KURATA et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34; EP 1 046 717 A9; JP 2001-286300 (P2001-286300A)).
A probe composed of a single-stranded oligonucleotide, wherein a portion other than the 3 ′ end of the probe, preferably the 5 ′ end side, more preferably the 5 ′ end or 5 ′ end is labeled with a fluorescent dye, and 3 ′ It is a probe that keeps the 3′OH group of terminal ribose or deoxyribose free. That is, the probe is used as a PCR primer (in the present invention, sometimes referred to as a plumer probe). The corresponding nucleic acid to be amplified is labeled with a fluorescent dye. This is a method for monitoring in real time a change in fluorescence intensity of a PCR measurement (reaction) system between a denatured amplified nucleic acid and a hybridized probe. In the same manner as described above, the concentration of the corresponding nucleic acid before amplification is determined from the change rate curve.
[0148]
II) Method of the present invention
This is a method using the nucleic acid probe described in the methods A and B of the present invention.
Since the structure, operation, and measurement are as described in the first invention and the above, they are omitted here.
Various nucleic acid amplification methods that can be suitably used in the second invention are as described above. Therefore, the method for amplifying a target nucleic acid and measuring the concentration or copy number of the target nucleic acid before amplification in the present invention is a method in which the principle of the present invention is applied to the nucleic acid amplification method.
[0149]
That is, in the nucleic acid amplification described above, the measurement system contains at least one kind of target nucleic acid and at least one kind of internal standard nucleic acid and contains a target nucleic acid probe or an internal standard nucleic acid probe, or a target nucleic acid probe and an internal standard. Fluorescence of the fluorescent dye labeled on the target nucleic acid probe caused by hybridization between the target nucleic acid and the target nucleic acid probe before and after the reaction in a measurement system containing at least two types in combination with the nucleic acid probe In this method, a change or amount of intensity is measured, and the concentration or copy number of the target nucleic acid before amplification is measured from the measured value and the amount of internal standard nucleic acid added.
[0150]
Below, the case where it applies to a real-time quantitative PCR method is illustrated.
The real-time quantitative PCR method of the present invention comprises adding a target nucleic acid probe or / and an internal standard nucleic acid probe to a measurement system containing at least one type of target nucleic acid and an internal standard nucleic acid at a known concentration, so that the target nucleic acid or / and the internal standard are added. It is characterized by amplifying a nucleic acid and monitoring (measuring) the change or amount of change in the fluorescence intensity of the measurement system before and after the reaction of the fluorescent dye labeled with each probe in the amplification process. In the real-time quantitative PCR of the present invention, as the target nucleic acid probe or the internal standard nucleic acid probe, any of the above-mentioned nucleic acid probes can be suitably used regardless of whether they are known or of the present invention. Any one may be used as long as it hybridizes with the target nucleic acid and its amplification product during the PCR cycle, preferably 10 to 25, particularly preferably 15 to 20. That is, a simple probe may be designed as either a forward type primer or a reverse type primer.
[0151]
As simple nucleic acid probes and primers, various nucleic acid probes used in the above-mentioned known methods and the nucleic acid probes described in the methods A and B of the present invention can be preferably used. The nucleic acid probe described in the methods A and B of the present invention is more preferable. Particularly preferred probes are those of v) to ix) of the method B of the present invention.
When used as a primer, it is not always necessary to label both the forward and reverse primers with a fluorescent dye. Since the present invention can be achieved, either one is sufficient.
Among the nucleic acid probes of the method B of the present invention, a nucleic acid probe in which the 3 'OH group of deoxyribose or ribose at the 3' end is not modified can be used as a primer. In this case, various nucleic acid probes used in known methods are also in the same form.
[0152]
When this primer is used, if the 3 ′ or 5 ′ end of the target nucleic acid probe or internal standard nucleic acid probe cannot be designed as G or C from the base sequence of the target nucleic acid or internal standard nucleic acid, 5 of the probe oligonucleotide Even if 5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine is added to the “terminal”, or 5′-guanylic acid or 5′-cytidylic acid is added to the 3 ′ terminal, the present invention The object can be suitably achieved. Therefore, in the present invention, the target nucleic acid probe and internal standard nucleic acid probe designed so that the base at the 3 ′ or 5 ′ end is G or C are the target nucleic acid and the probe designed from the base sequence of the internal standard nucleic acid. In addition, 5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine is added to the 5 ′ end of the probe, and 5′-guanylic acid or 5′-cytidylic acid is added to the 5 ′ end. It is defined as including an added probe.
[0153]
When a target nucleic acid probe or an internal standard nucleic acid probe is used as a primer for PCR, a specific example of a method for measuring a target nucleic acid is described in a data analysis method (described later) obtained by real-time quantitative PCR. I will omit it.
[0154]
Of the nucleic acid probes, nucleic acid probes modified with deoxyribose at the 3 ′ end or the 3′OH group of ribose cannot be used as primers, but the probe is used in a PCR reaction system together with a reverse primer and a forward primer. It can be added and PCR can be performed. In this case, during the nucleic acid extension reaction, the target nucleic acid probe or internal standard nucleic acid probe hybridized to the target nucleic acid or amplified target nucleic acid or internal standard nucleic acid or amplified internal standard nucleic acid is decomposed by the polymerase and decomposed and removed from the nucleic acid probe complex. Is done. That is, it is used as a simple probe. However, in the probe, the base sequence region that can hybridize to the target nucleic acid and the internal standard nucleic acid is between the regions that can hybridize to both the primers.
[0155]
At this time, the change or amount of the fluorescence intensity of the dye that has labeled the target nucleic acid probe is measured (as an example, the fluorescence intensity of the reaction system is measured). In addition, the fluorescence intensity (value) of the dye labeling the target nucleic acid probe when the target nucleic acid or the amplified target nucleic acid or the internal standard nucleic acid or the amplified internal standard nucleic acid is hybridized with each probe (as an example, The former can be an annealing reaction, a reaction system during a nucleic acid extension reaction until the probe is removed from the target nucleic acid probe complex by polymerase, and the latter can be a fluorescence intensity value of a reaction system in which a nucleic acid denaturation reaction has been completed. ).
[0156]
When measuring the change or amount of fluorescence intensity of the internal standard nucleic acid probe, the hybridization reaction is performed on the internal standard nucleic acid at various concentrations, and the change or amount of change of the fluorescence intensity is measured. This measurement is performed in real time for each cycle.
For each concentration of internal standard nucleic acid, the amount of change (rate) in fluorescence intensity (value) is calculated for each cycle for the fluorescent dye labeled on the internal standard nucleic acid probe.
[0157]
Next, a relational expression of the concentration before amplification of the internal standard nucleic acid is obtained from these change amounts (rates) (for example, a calibration curve of the change amount (rate) as a function of the concentration before amplification of the internal standard nucleic acids is prepared. (Similar to the data analysis method when the target nucleic acid probe is used as a primer.) In general, the internal standard nucleic acid concentration and the amount of change are linearly proportional to each other in a specific number of cycles, so that the cycle can be found by performing measurement in real time.
Next, the same amount of change (rate) in the number of cycles is obtained for the target nucleic acid. This amount of change (rate) is applied to the calibration curve or the relational expression. Thus, the target nucleic acid can be measured. A specific example is shown in the examples.
[0158]
When a nucleic acid probe is used as a primer, the Tm value of the probe-nucleic acid complex when the target nucleic acid probe is hybridized with the target nucleic acid or the internal standard nucleic acid probe is hybridized with the nucleic acid complex of the primer. It is desirable that the base sequence of each target nucleic acid probe is designed so that the Tm value is within a range of ± 15 ° C., preferably ± 5 ° C.
[0159]
As the PCR reaction of the present invention proceeds, each amplified nucleic acid is secondarily labeled with a fluorescent dye useful in the practice of the present invention. Therefore, the fluorescence intensity value of the reaction system in which the nucleic acid denaturation reaction has been completed is large or small, but in the reaction system in which the annealing reaction is completed or in the nucleic acid extension reaction, the fluorescence intensity of the reaction system is the former fluorescence intensity. Decrease or increase from strength.
[0160]
The PCR reaction can be performed under the same reaction conditions as in a normal PCR method. In particular, in the nucleic acid probes A and B of the present invention, nucleic acid amplification can be performed in a reaction system in which the Mg ion concentration is low (1 to 2 mM). Of course, it can also be carried out in a reaction system in the presence of a high concentration (2 to 4 mM) of Mg ions used in conventionally known quantitative PCR.
[0161]
The third invention of the present invention is the above-described nucleic acid measurement method (first invention), nucleic acid amplification method (second invention), polymorphism measurement / analysis / analysis method (described later), and various measurements / measurements such as FISH. It is an invention of a method of analyzing data obtained when an analysis / analysis method (described later) or the like is performed, or when various methods are performed using various devices (described later).
Hereinafter, for simplification, the real-time quantitative PCR method will be described among nucleic acid amplification methods, but data may be analyzed according to other methods as well.
[0162]
The real-time quantitative PCR method is currently a computer-readable record in which each procedure of a reaction device for performing PCR, a device for detecting luminescence of a fluorescent dye, a user interface, that is, a data analysis method is programmed and recorded. It is measured in real time on a medium (also known as Sequence Detection Software System) and an apparatus composed of a computer that controls and analyzes the data. Therefore, the measurement of the present invention is also performed with such an apparatus.
[0163]
First, a real-time quantitative PCR analyzer will be described below. The apparatus used in the present invention may be any apparatus as long as it can monitor PCR in real time. For example, ABI PRISM^TM 7700 Sequence Detection System SDS 7700 (Perkin Elmer Applied Biosytems, USA), light cycler^TM  A system (Roche Diagnostics Inc., Germany) can be cited as a particularly suitable one.
[0164]
The PCR reaction apparatus is an apparatus that repeatedly performs a heat denaturation reaction, annealing reaction, and nucleic acid extension reaction of a target nucleic acid. The detection system consists of an argon laser for fluorescence excitation, a spectrograph, and a CCD camera. Further, a computer-readable recording medium on which each procedure of the data analysis method is programmed and recorded is used by being installed in the computer, and the system is controlled via the computer and output from the detection system. A program for analyzing data is recorded.
[0165]
The data analysis program recorded on the computer-readable recording medium is a process for measuring the fluorescence intensity for each cycle, and the measured fluorescence intensity is displayed on the computer display as a function of the cycle, that is, as an amplification plot of PCR. The process of displaying, the process of calculating the PCR cycle number (threshold cycle number: Ct value) where fluorescence intensity starts to be detected, the process of creating a calibration curve for obtaining the copy number of the sample nucleic acid from the Ct value, the data of each process, plot The process of printing values. When PCR progresses exponentially, a linear relationship is established between the concentration or copy number (Log value) of the target nucleic acid at the start of PCR and the amount of change (rate) or Ct value of fluorescence intensity. Therefore, a calibration curve is prepared in advance using the known concentration or copy number of the target nucleic acid and the internal standard nucleic acid, and the amount of change in fluorescence intensity for a sample containing the target nucleic acid of unknown concentration or copy number ( Ratio) or Ct value, the concentration or copy number of the target nucleic acid before nucleic acid amplification can be calculated.
[0166]
The characteristics of the method for analyzing the data obtained by the real-time quantitative PCR method as described above will be described below.
The first feature is that in the method of analyzing data obtained by the real-time quantitative PCR method, the nucleic acid to be analyzed in each cycle (in addition to the target nucleic acid, an internal standard nucleic acid is also included here). ) To the nucleic acid probe corresponding to the nucleic acid (specifically hybridizes), the fluorescence intensity value of the fluorescent dye that labeled the probe is determined from the probe / nucleic acid complex in each cycle by polymerase. This is an arithmetic processing step of correcting by the fluorescence intensity value of the dye when the probe is decomposed and the fluorescent dye is liberated or when the complex is dissociated by a nucleic acid denaturation reaction, that is, a correction arithmetic processing step. In the present invention, “the nucleic acid to be analyzed and the nucleic acid probe corresponding to (specifically hybridizes with) the nucleic acid” are an internal standard nucleic acid and an internal standard nucleic acid probe, and a target nucleic acid and a target nucleic acid probe ( The same applies hereinafter).
[0167]
“The fluorescence intensity value of the fluorescent dye labeled with the probe when the amplified nucleic acid to be analyzed is hybridized to the nucleic acid probe corresponding to (specifically hybridizes with) the nucleic acid” is specifically exemplified Then, when the probe is merely used as a nucleic acid probe, the fluorescence intensity value of the fluorescent dye that has labeled the nucleic acid probe in the annealing reaction system at 40 to 85 ° C., preferably 50 to 80 ° C. in each cycle of PCR. (More specifically, the fluorescence intensity value of the reaction system measured at a measurement wavelength specific to the dye) (hereinafter the same). And it means a reaction system in which the reaction is completed. When the probe is used as a primer, it is an annealing reaction system or a nucleic acid extension reaction system at 40 to 85 ° C., preferably 50 to 80 ° C. The actual temperature depends on the length of the amplified nucleic acid.
[0168]
In addition, “the fluorescence intensity value of the dye when the probe is decomposed by the polymerase and the fluorescent dye is released from the dissociation of the lobe / nucleic acid complex or the complex is dissociated by the nucleic acid denaturation reaction” The reaction system for heat denaturation of nucleic acid in each cycle of PCR, specifically, when the reaction temperature is 90 to 100 ° C., preferably 94 to 96 ° C., and the target nucleic acid probe dye in the reaction system where the reaction is completed Examples of the fluorescence intensity value of the reaction system when measured at the measurement wavelength involved (the same applies hereinafter). In this case, when the probe is merely used as a nucleic acid probe, a reaction system in which the fluorescent dye is released by decomposing the probe by a polymerase from the probe / nucleic acid complex is also included.
[0169]
The correction calculation process in the correction calculation process may be any process that meets the object of the present invention. Specifically, the correction calculation process includes a process according to the following [Equation 1] or [Equation 2]. Things can be illustrated.
f_n= F_{hyb, n}/ F_{den, n}          [Formula 1]
f_n= F_{den, n}/ F_{hyb, n}          [Formula 2]
[Where,
f_n: Correction calculation processing value in the n-th cycle calculated by [Equation 1] or [Equation 2],
f_{hyb, n}: In the n-th cycle, the fluorescence intensity value of the reaction system when the amplified nucleic acid to be analyzed is hybridized with the nucleic acid probe corresponding to the nucleic acid,
f_{den, n}: In the n-th cycle, when the probe is decomposed by the polymerase from the probe-nucleic acid complex and the fluorescent dye is released, or when the probe-nucleic acid complex is dissociated by the nucleic acid denaturation reaction, (Fluorescence intensity value)
In this process, the correction calculation processing value obtained by the above processing is displayed on the computer display and / or the value is similarly displayed and / or printed in the form of a graph as a function of each cycle number. Includes steps.
[0170]
The second feature is that the correction calculation processing value according to [Equation 1] or [Equation 2] in each cycle is substituted into the following [Equation 3] or [Equation 4], and the amount of fluorescence change (fluorescence change rate) between the samples. Alternatively, it is a data analysis method that calculates (fluorescence change rate) and compares them.
[0171]
F_n= F_n/ F_a          [Formula 3]
F_n= F_a/ F_n          [Formula 4]
[Where,
F_n: Fluorescence change amount (fluorescence change rate or fluorescence change rate) calculated by [Formula 3] or [Formula 4] in the n-th cycle,
f_n: Correction calculation processing value according to [Formula 1] or [Formula 2] in the n-th cycle
f_a: Correction calculation processing value according to [Formula 1] or [Formula 2], f_nOf any number of cycles before the change is observed, but typically for example 10 to 40 cycles, preferably 15 to 30 cycles, more preferably 20 to 30 cycles Is adopted. ]
[0172]
In this process, the calculated value obtained in the above process is displayed and / or printed on a computer display, or the comparison value or the value is similarly displayed in graph form as a function of each cycle number. Alternatively, although it includes a sub-step for printing, the sub-step may or may not be applied to the correction calculation processing value according to [Formula 1] or [Formula 2].
[0173]
The third feature is
(3.1) [Formula 5], [Formula 6] or [Formula 7] using the data of the fluorescence change amount (fluorescence change rate or fluorescence change rate) calculated by [Formula 3] or [Formula 4] The process of processing by
log_b(F_n), Ln (F_n[Formula 5]
log_b{(1-F_n) × A}, ln {(1-F_n) × A} [Formula 6]
log_b{(F_n−1) × A}, ln {(F_n−1) × A} [Formula 7]
[0174]
[Where,
A, b: Any numerical value, preferably an integer value, more preferably a natural number. When A = 100 and b = 10, {(F_n−1) × A} is expressed as a percentage (%).
F_n: [Fluorescence change amount in n cycles calculated by [Equation 3] or [Equation 4] (fluorescence change rate or fluorescence change rate)],
(3.2) An arithmetic processing step for obtaining the number of cycles in which the arithmetic processing value of (3.1) has reached a certain value;
(3.3) Arithmetic processing for calculating a relational expression between the number of cycles in a sample containing a target nucleic acid and / or an internal standard nucleic acid at a known concentration and the concentration or copy number of the target nucleic acid and internal standard nucleic acid before nucleic acid amplification at the start of the reaction process,
(3.4) A calculation process for obtaining the concentration or copy number of the target nucleic acid before nucleic acid amplification in the sample containing the target nucleic acid,
Is a data analysis method. A process consisting of (3.1) → (3.2) → (3.3) → (3.4) is preferable.
[0175]
In each of the processes (3.1) to (3.3), the processing value obtained by each processing is displayed on a computer display and / or the value is expressed in the form of a graph as a function of the number of cycles. Similarly to the above, it may include a sub-step of displaying and / or printing. Since the arithmetic processing value obtained in the process (3.4) needs to be printed at least, the process includes a sub-step for printing. The arithmetic processing value obtained in the above (3.4) may be further displayed on a computer display.
It should be noted that the correction processing value according to [Formula 1] or [Formula 2] and the calculation processing value according to [Formula 3] or [Formula 4] are displayed on the computer display in the form of a graph as a function of the number of cycles. Since it may or may not be printed, the display and / or printing sub-steps may be added as necessary.
[0176]
In the above method, the reaction system for measuring the internal standard nucleic acid and the reaction system for measuring the target nucleic acid may be combined, or may be separated.
The data analysis method is particularly effective when the real-time quantitative PCR method measures the amount of decrease in the emission of the fluorescent dye.
[0177]
The data analysis method of the present invention has the above-described characteristics, but in actual analysis, it is preferable to perform the following procedure.
1) A reaction system that is regarded as a substance that inhibits a hybridization reaction and / or a nucleic acid amplification reaction between a nucleic acid and a nucleic acid probe, or a polymorphic nucleic acid, and has first to third characteristics of the present invention. Perform all or up to one of the processes.
2) Next, for an unknown sample (which is assumed to contain a target nucleic acid) in which the inhibitor or polymorphic nucleic acid is present, a nucleic acid probe, various concentrations of internal standard nucleic acid, and internal standard nucleic acid probe A nucleic acid amplification reaction is performed using In this case, a calibration curve is obtained for the internal standard nucleic acid, but not for the target nucleic acid.
[0178]
3) The processed data for the target nucleic acid and the internal standard nucleic acid obtained in 1) and 2) are compared and examined.
(1) When the processed data for the internal standard nucleic acid (data processed by the above data analysis method; the same applies hereinafter) is the same or substantially the same, the inhibitor or polymorphism is contained in the sample. It is regarded as not. Therefore, various graphs, relational expressions, and calibration curves for the target nucleic acid and the internal standard nucleic acid obtained in 1) can be used for the unknown sample.
[0179]
(2) If the processed data for the internal standard nucleic acid is not the same or substantially the same, it is considered that the inhibitor or polymorphism is contained in the sample. The rate of change, that is, the coefficient of change of various graphs, relational expressions, and calibration curves for the internal standard nucleic acid obtained in 1) and 2) is calculated.
(3) The change coefficient is applied to various graphs, relational expressions, and calibration curves for the target nucleic acid obtained in 1).
The various graphs, relational expressions, and calibration curves thus obtained are graphs, relational expressions, and calibration curves for obtaining the concentration or copy of the target nucleic acid before target nucleic acid amplification.
[0180]
There are various methods for obtaining the concentration or copy number before amplification of the target nucleic acid of the present invention, and any method can be adopted as long as the object of the present invention is achieved. For example, it was shown in Example 1.
Further, as a simple method, an internal standard nucleic acid is added to a sample that is considered to contain the inhibitory substance, and the target nucleic acid probe and / or the internal standard nucleic acid probe of the present invention is used or not used. Then, the target nucleic acid and the internal standard nucleic acid in the sample are amplified by the real-time monitoring quantitative PCR method. For each amplified nucleic acid, a hybridization reaction is performed using the target nucleic acid probe and the internal standard nucleic acid probe of the present invention. The amount of change in fluorescence intensity of the fluorescent dye labeled on each probe is measured. Of course, when the amount of change is obtained, it is preferable to use the data analysis method of the present invention. The relationship between the amount of change for the internal standard nucleic acid and the concentration of the internal standard nucleic acid is considered to be applicable to the target nucleic acid (in addition, in a system in which there are no inhibitors or polymorphisms in the hybridization reaction and / or nucleic acid amplification reaction). Make sure that you can make such an assessment). Therefore, a relational expression between the change amount of the internal standard nucleic acid and the internal standard nucleic acid concentration is obtained in advance using the known sample.
The concentration or copy number of the amplified target nucleic acid and internal standard nucleic acid can be determined from the relational expression. Since the concentration or copy number of the internal standard nucleic acid before amplification is known, the concentration or copy number of the amplified target nucleic acid before amplification can be estimated.
[0181]
A fourth feature of the real-time quantitative PCR analyzer is a real-time quantitative PCR measurement and / or analysis device having an operation and storage means for executing the data analysis method for the real-time quantitative PCR method of the present invention. It is.
In this case, since the measurement system contains at least one or more target nucleic acids, a measurement apparatus that can measure at least one or more wavelengths is preferable. Moreover, it is more preferable if it is an apparatus that can excite a fluorescent dye with at least one or more wavelengths.
[0182]
A fifth feature of the present invention is that in the computer-readable recording medium in which each procedure of the data analysis method for analyzing PCR using a real-time quantitative PCR analyzer is programmed and the program is recorded, the data analysis of the present invention is performed. A computer-readable recording medium recording a program that allows a computer to execute each procedure of the method.
[0183]
The fourth invention of the present invention is an invention in which the above-mentioned various methods are applied to a method for analyzing or measuring a polymorphism (including SNP) or / and mutation of a target nucleic acid.
Further, the measurement kit, the data analysis method for the measurement, the measurement apparatus, and the computer-readable recording medium recording the procedure for causing the computer to execute the data analysis process as a program.
[0184]
When analyzing data obtained by a method for analyzing or measuring polymorphism or / and mutation of the target nucleic acid of the present invention, when the nucleic acid to be analyzed is hybridized with a corresponding nucleic acid probe A process of correcting the fluorescence intensity value of the fluorescent dye labeled with the probe with the fluorescence intensity value of the fluorescent dye labeled with the probe when the above-mentioned probe is not hybridized. The obtained data is highly reliable (application of data analysis method).
[0185]
The fourth invention is a measuring apparatus for analyzing or measuring polymorphism or / and mutation of a target nucleic acid, and having a means for processing data obtained by the data analysis method. .
In addition, a computer-readable recording of a procedure for causing a computer to execute the above-described process of correcting data obtained by a method for analyzing or measuring polymorphism or / and mutation of a target nucleic acid. It is a recording medium.
[0186]
The fifth invention of the present invention is an invention in which the novel nucleic acid measurement method of the present invention is applied to various nucleic acid measurement methods, for example, FISH method, LCR method, SD method, TAS method and the like.
An example is given below.
When applying to FISH method
That is, the method of the present invention can suitably use a complex microbial or symbiotic microbial cell or a cell homogenate as a sample for nucleic acid measurement. The complex microbial system or the symbiotic microbial system is a substance that inhibits the hybridization reaction of the present invention (including, of course, the hybridization of an internal standard nucleic acid and its internal standard nucleic acid probe) and / or a nucleic acid amplification reaction, This is because a substance that inhibits light emission of the fluorescent dye of the probe may be included.
[0187]
The above-described novel nucleic acid measurement method, nucleic acid amplification method, and
A complex microbial system or a symbiotic microbial system using one or more of data analysis methods, measurement apparatuses (including those incorporating a computer-readable recording medium) and various devices (described later) By quantifying the number of copies of 5SrRNA, 16SrRNA, 23SrRNA or their gene DNA of a specific strain in, the abundance of the specific strain in the system can be measured. This is because the copy number of 5S rRNA, 16S rRNA or 23S rRNA gene DNA is constant depending on the strain.
[0188]
The sixth invention of the present invention includes reaction liquids or measurement kits and devices (for example, the following DNA chip, etc.) that can be conveniently used when carrying out the novel nucleic acid measurement method of the present invention. ).
The reaction liquids or measurement kits include at least one internal standard nucleic acid, at least one target nucleic acid probe or a target nucleic acid primer probe and / or at least one internal standard nucleic acid probe or an internal standard nucleic acid primer probe. Preferably, other components (buffer solution, trace metal ions, etc.) are appropriately contained. However, since these components can be used by adding to the measurement system at the time of use, the reaction liquids or measurement kits are not limited to these components. The form may be either dry or liquid. In the reaction solutions or measurement kits in the second invention of the present invention (nucleic acid measurement method using nucleic acid amplification reaction), other components include primers (when no primer probe is used), nucleic acid amplification such as polymerase, etc. It is preferred to include reagents necessary for the reaction.
[0189]
Any device can be used as long as it can carry out the method of the present invention, and examples thereof include the following DNA chips.
That is, a plurality of at least one internal standard nucleic acid probe and / or at least one target nucleic acid probe that can be used in the present invention are bound to the surface of a solid support. At least one target nucleic acid is measured by hybridizing the internal standard nucleic acid and measuring the change or amount of change in fluorescence intensity (before and after hybridization) specific to the fluorescent dye of the nucleic acid probe derived from each probe / nucleic acid complex It is a device that can measure the amount or the concentration of
[0190]
In the device, at least one type of target nucleic acid probe and / or at least one type of internal standard nucleic acid probe is arrayed and bound to the surface of the solid support, and each nucleic acid probe bound to the surface of the solid support. In this device, at least one temperature sensor and a heater are installed on the opposite surface, and the temperature can be adjusted so that the nucleic acid probe binding region is in an optimum temperature condition.
[0191]
That is, the target nucleic acid probe and the internal standard nucleic acid probe of the present invention can be immobilized on a solid (support layer) surface, for example, the surface of a slide glass. This form is now called a DNA chip. Can be used for gene expression monitoring, base sequence determination, mutation analysis, polymorphism analysis such as single nucleotide polymorphism (SNP), etc. . Of course, it can also be used as a nucleic acid measuring device (chip).
Therefore, the fifth invention of the present invention is a method for achieving these items in addition to the measurement of the target nucleic acid.
[0192]
In the present invention, by preparing a device in which many target nucleic acid probes and / or internal standard nucleic acid probes having different base sequences are individually bound on the same solid surface, multiple types of target nucleic acids can be measured simultaneously. Therefore, since the target nucleic acid can be measured in exactly the same manner as in the case of the DNA chip, it is also a new DNA chip. Under optimal reaction conditions, nucleic acids other than the target nucleic acid do not hybridize to the target nucleic acid probe, so that the amount of fluorescence emission is not changed. Therefore, it is not necessary to wash the nucleic acid that does not specifically hybridize to the target nucleic acid probe.
[0193]
The basic operation of a novel method for measuring nucleic acid using the device of the present invention is to place a solution containing a nucleic acid to be analyzed on a solid surface to which a target nucleic acid probe corresponding to the nucleic acid to be analyzed is bound, to the nucleic acid to be analyzed, and to the nucleic acid to be analyzed. Simply hybridize the corresponding target nucleic acid probe.
As a result, the fluorescence intensity of the fluorescent dye labeled with the target nucleic acid probe changes before and after hybridization according to the amount of nucleic acid to be analyzed, and the nucleic acid to be analyzed can be measured from the change amount (rate) of the fluorescence intensity. Become.
[0194]
In actual measurement, first, using the internal standard nucleic acid and the corresponding internal standard nucleic acid probe, the amount of change in fluorescence intensity (rate) of the fluorescent dye labeled with the internal standard nucleic acid probe and the amount of internal standard nucleic acid or Create a relational expression or calibration curve with concentration. By applying the change amount (rate) of fluorescence intensity related to the target nucleic acid to this, the target nucleic acid can be measured. This operation is as described above.
[0195]
Furthermore, in the device of the present invention, by controlling the temperature for each target nucleic acid probe with a micro heater, it is possible to control the optimum reaction conditions for each probe, so that accurate concentration measurement is possible. In addition, the dissociation curve between the target nucleic acid probe of the present invention and the target nucleic acid can be analyzed by continuously changing the temperature with a micro heater and measuring the amount of fluorescence during that time. Based on the difference in the dissociation curve, the properties of the hybridized nucleic acid can be determined and the SNP can be detected.
[0196]
The specific conditions for measurement are described below.
These methods can be carried out by conventional known methods (Analytical Biochemistry, 183, 231-244, 1989; Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63 1143-1147, 1997).
[0197]
The hybridization conditions are, for example, a salt concentration of 0 to 2 molar, preferably 0.1 to 1.0 molar, and a pH of 6 to 8, preferably 6.5 to 7.5.
The reaction temperature is preferably within the range of Tm value ± 10 ° C. of the target nucleic acid probe complex that is a product of the hybridization reaction. By doing so, non-specific hybridization can be prevented. When it is lower than Tm-10 ° C., nonspecific hybridization occurs, and when it exceeds Tm + 10 ° C., hybridization does not occur. The Tm value can be obtained in the same manner as the experiment necessary for designing the target nucleic acid probe.
[0198]
The reaction time is 1 second to 180 minutes, preferably 5 seconds to 90 minutes. When the time is less than 1 second, hybridization is not sufficiently completed. Also, there is no particular meaning if the reaction time is too long. The reaction time is greatly influenced by the nucleic acid species, that is, the length of the nucleic acid or the base sequence.
[0199]
The concentration of the nucleic acid to be analyzed in the reaction solution is preferably 0.1 to 10.0 nM. The concentration per spot of the target nucleic acid probe corresponding to the analyte in the device of the present invention is preferably 1.0 to 25.0 nM.
When preparing a calibration curve or a relational expression for the internal standard nucleic acid, it is desirable to use the internal standard nucleic acid of the present invention at a ratio of 0.4 to 1.0 with respect to the internal standard nucleic acid probe.
The devices of the present invention include reaction liquids or measurement kits that can perform the method of the present invention using the devices. The reaction solutions or measurement kits contain at least one internal standard nucleic acid. Other components are the same as those in the reaction solutions or measurement kits.
[0200]
The seventh aspect of the method of the present invention is a method for separating, recovering and concentrating a target nucleic acid, which enables the above-described methods of the present invention to be carried out simply, rapidly, accurately and specifically.
This method is a target nucleic acid separation / separation method in which all nucleic acids containing the target nucleic acid are cleaved by at least one restriction enzyme that does not cleave the target nucleic acid base sequence region, and then only the nucleic acid fraction containing the target nucleic acid base sequence is separated and recovered. This is a recovery and concentration method. For example, preferably, a method comprising the following means may be adopted. However, the method of the present invention is not limited by this example.
(1) A nucleic acid such as a genome containing a target nucleic acid base sequence region is treated with at least one restriction enzyme.
(2) The processed product is subjected to molecular fractionation.
Further, it is preferable to add the following means.
(3) Concentrate the molecular fraction.
[0201]
The restriction enzyme is not particularly limited as long as it does not cleave the target base sequence region. That is, it may be changed according to the purpose. A well-known thing can be utilized suitably. For example, Bfa I, Bsa JI, Bss KI, Dde1, Mse I, Bso FI, Hha I, Hph I, Mnl I, Rca I, Alu I, Msp I and the like can be mentioned. It is preferable to use these in appropriate combinations according to the purpose. However, the present invention is not limited by these.
The restriction enzyme treatment conditions may be in accordance with the description (protocol) of the instructions attached to the restriction enzyme reagent kit.
What is necessary is just to employ | adopt an appropriate thing for the said molecular fraction according to the objective for the purpose. For example, molecular fractionation methods such as filtration methods using various filters, gel filtration methods using various fillers (including HPLC methods, etc.) can be mentioned.
The concentration method may be a normal method according to the purpose. Examples thereof include a concentration method using a solvent such as ethanol, freeze drying, and air drying.
[0202]
The seventh invention of the method of the present invention is a simple method for preparing a high-purity artificial gene (double-stranded DNA) of 50 bp or more having an arbitrary base sequence.
The preparation method includes the following means.
1) A 50 bp or more single-stranded oligonucleotide having an arbitrary base sequence is artificially synthesized using a DNA synthesizer.
2) Nucleic acid amplification reactions are performed by various gene amplification methods using a synthesized single-stranded oligonucleotide of 50 bp or more as a template.
[0203]
Any DNA synthesizer may be used as long as it achieves the object of the present invention. Preferably, a DNA synthesizer (ABI394) (Perkin Elmer Japan Applied) may be used. The gene amplification method may be any method as long as the object of the present invention is achieved. Preferably, a PCR method may be used.
In order to prevent the risk that single-stranded oligonucleotides other than the target will be mixed into the gene amplification product, it is desirable to use the template after sufficiently diluting it. The dilution rate of this template is not particularly limited. Further, by diluting the gene amplification product and again performing gene amplification using the diluted solution as a template, it is possible to eliminate the risk of contamination by single-stranded oligonucleic acid other than the above-mentioned purpose. The number of times this gene amplification is repeated may be determined according to the purpose of the present invention, and is not particularly limited.
[0204]
The first to eighth inventions of the present invention can be used in various fields such as medicine, forensic medicine, anthropology, ancient biology, biology, genetic engineering, molecular biology, agriculture, plant breeding. Also referred to as a complex microbial system or a symbiotic microbial system, such as microbial systems in which various types of microorganisms are mixed, or at least one type of microorganism is mixed with cells from other animals and plants and cannot be isolated from each other. It can be suitably used for analysis and analysis. The microorganism referred to herein is a microorganism generally referred to, and is not particularly limited.
[0205]
【Example】
Next, the present invention will be described more specifically with reference to examples.
Example 1
As the target nucleic acid (gene), the nec1 gene, which is considered to be a causative gene for potato scab, was used to quantify the nec1 gene from soil.
A) Various methods
1) Synthesis method of oligodeoxyribonucleotide (nucleic acid):
Except where otherwise specified, oligodeoxyribonucleotides (hereinafter referred to as oligonucleotides) were synthesized using a DNA synthesizer (ABI394) (Perkin Elmer Japan Applied Co., Ltd.).
[0206]
2) Labeling of oligonucleotides with fluorescent dyes
(1) Modification of oligonucleotide with linker
Using a 5'Amino-Modifier C6 kit (Glen Research, USA), the 5'-phosphate group of the 5'-terminal nucleotide is linked to a linker- (CH₂)₆Modified with -SH. Using this modified nucleotide, an oligonucleotide in which the phosphate group at the 5 'end was modified with the linker was synthesized using a DNA synthesizer (ABI394). DNA synthesis was performed by β-cyanoethyl phosphoramidite method and 5′-end TrON method. After the synthesis, the protecting group was removed with 28% aqueous ammonia at 55 ° C. for 5 hours.
[0207]
(2) Purification of the synthesized product:
The synthetic oligonucleotide obtained above was dried to obtain a dried product. 0.5M NaHCO_Three/ Na₂CO_ThreeDissolved in buffer (pH 9.0). The lysate was subjected to gel filtration with a NAP-10 column (Pharmacia) to remove unreacted substances.
[0208]
(3) Dye labeling:
The gel filtrate was dried and dissolved in 150 μL of sterilized water (oligonucleotide solution). 1 mg of a fluorescent dye (for example, BODIPY FL) -Chloride (Molecular Probes, USA) is dissolved in 100 μL of DMF, and the oligonucleotide solution, 1M NaHCO 3 is dissolved._Three/ Na₂CO_ThreeAfter adding 150 μL of the buffer and stirring, the reaction was allowed to proceed overnight at room temperature.₂)₆A fluorescent dye was bound to -SH.
[0209]
(4) Purification of the synthesized product:
The reaction product was subjected to gel filtration with NAP-25 (Pharmacia) to remove unreacted material. Reversed-phase HPLC using SEP-PAC C18 column was performed, and the oligonucleotide linker- (CH₂)₇-NH₂The target product in which a fluorescent dye was bound to was fractionated. The fraction was gel filtered with NAP-10 (Pharmacia). Thus, an oligonucleotide having a fluorescent dye bound to the 5 'end was obtained.
[0210]
(5) Quantification of oligonucleotide with fluorescent dye bound to 5 'end of the present invention
The measurement was carried out by measuring a value of 260 nm with a spectrophotometer. Further, as a result of performing scanning with an absorbance of 650 nm to 220 nm using a spectrophotometer, it was confirmed that the probe had absorption of a fluorescent dye and DNA. Furthermore, as a result of conducting a purity test of the purified product by reverse-phase HPLC as described above, it was confirmed that it was a single peak.
[0211]
(6) Reversed phase chromatography
In addition, the conditions of said reverse phase chromatography are as follows.
・ Elution solvent A: 0.05N TEAA 5% CH_ThreeCN
・ Elution solvent B (for gradient): 0.05N TEAA 40% CH_ThreeCN
・ Column: SEP-PAK C18; 6 × 250mm
・ Elution rate: 1.0ml / min
・ Temperature: 40 ℃
・ Detection: 254nm
[0212]
3) Method for phosphorylating the 3'-position OH group at the 3 'end
During the synthesis of the oligonucleotide, a 3 'phosphate group was generated by a deprotection reaction (Glen Research catalog number 20-2913).
4) Agarose electrophoresis method
Performed in the usual way. That is,
・ Agarose concentration: 0.5%
・ Buffer: TBE buffer, pH9.3
・ Temperature: Room temperature
[0213]
5) Preparation of internal standard nucleic acid (DNA gene)
Primers NEC-F and NEC-R, NECM-F and NECM-R comprising oligonucleotides having the base sequences shown in Table 1 were prepared. Using these primers, mutation was introduced into the target nucleic acid by the overlap extention PCR method (guideline for using the PCR method, pages 29 to 37, Chugai Medical, 1998). F means forward primer and R means reverse primer.
[0214]

[0215]
In addition, FIG. 1 shows to which region of the nec1 gene the oligonucleotide having each base sequence in Table 1 hybridizes (corresponds).
In addition, the base sequence of NECM-R is obtained by mutating base sequences 11, 12, 13, and 15 (from the 5 ′ end) in the table with respect to the sequence of the target nucleic acid (gene) (below) Table 2). Similarly, the base sequence of NECM-F is obtained by mutating the 11, 13, 14, and 15th bases (from the 5 'end) in the table (see Table 2 below).
[0216]

[0217]
The operation procedure is as follows.
(1) PCR was performed using Streptomyces turgidiscabies IFO16080 genomic DNA (purchased from IFO) as a template, a and d primers as one set, and b and c primers as one set. Genomic DNA was prepared as follows.
(2) The product was subjected to agarose gel electrophoresis. Each band corresponding to the amplified nucleic acid was cut out, and the amplified nucleic acid was extracted.
(3) Each extract was mixed and PCR was performed without adding new primers.
(4) The product was subjected to electrophoresis in the same manner as in 2) above. Only the band corresponding to the product having a length from A to B was cut out, and the amplification product was extracted.
(5) PCR was performed using the extract as a template and primers a and b as a set.
(6) The product was subjected to electrophoresis in the same manner as in 2) above. Only the band corresponding to the length from A to B was cut out again.
(7) Mutation insertion was confirmed with a sequencer. In addition, the amount of DNA was quantified.
[0218]
6) Preparation of target nucleic acid probe and internal standard nucleic acid probe
NECB-24 shown in Table 2 is the base sequence of the target nucleic acid probe, and NECMB-24 is that of the internal standard nucleic acid probe. In this example, the target nucleic acid probe is called NECB-24, and the internal standard nucleic acid probe is called NECMB-24.
NECB-24 was labeled with BODIPY FL by the method described above, and NECMB-24 was labeled with 6-TAMRA at the 5 'end by the method described above. Both were used by phosphorylating the 3 'OH group at the 3' end.
In the table, the 10, 11, 12 and 14th bases C, T, G and A of NECB-24 are mutated to A, G, C and T, respectively, in NECMB-24.
[0219]
7) PCR was performed under the following conditions.
-Denaturation reaction: 95 ° C, 15 sec
・ Annealing and detection: 62 ℃, 18sec
・ Extension: 72 ℃, 14sec
-Taq polymerase: Gene Taq (Nippon Gene Co., Ltd.)
Primer addition concentration: 500 nM, e and f (see FIG. 1 and Table 1)
・ Probe addition concentration: 50 nM
Template nucleic acid (Template): A mixture of PCR fragments of internal standard nucleic acid NECM1 and target nucleic acid NECB1 (PCR amplification product: about 700 bp with a and b plumers as a set)
Measurement device: Smart cycler system (Takara Bio Inc.) (hereinafter referred to as smart cycler for convenience).
[0220]
・ Channels used:
Channel 1 (Ex, 450-495 nm; Em, 505-537 nm).
Channel 3 (Ex, 527-555 nm; Em, 565-605 nm).
The fluorescence intensity of BODIPY FL was measured in channel 1, and the fluorescence intensity of 6-TAMRA was measured in channel 3 at the same time.
[0221]
8) Extraction of DNA from soil
A target nucleic acid was extracted from soil using Bio101 kit (for soil), and further purified using Wizard DNA Clean-Up system was used as a soil sample (sample).
[0222]
B) Experimental example
1) Evaluation of fabricated probe
A fluorescence intensity change rate (decrease) and a dissociation curve were prepared when the target nucleic acid probe NECB-24 and the internal nucleic acid probe NECMB-24 were hybridized with corresponding nucleic acids (target nucleic acid or internal standard nucleic acid). The same reaction buffer as that used in the PCR reaction was used.
The results are shown in FIGS. Both probes had a maximum fluorescence intensity change rate of about 70%, indicating a reasonable change rate. With NECB-24, the difference in Tm between the target nucleic acid and the internal standard nucleic acid is a little less than 20 ° C, and if it is detected at around 62 ° C, it becomes clear that only the target nucleic acid can be detected without detecting the internal standard nucleic acid. It was. Similarly, in NECMB-24, the difference in Tm value between the internal standard nucleic acid and the target nucleic acid is a little less than 20 ° C, and if it is detected at around 62 ° C, only the target nucleic acid can be detected without detecting the target nucleic acid. It became clear. Therefore, it was decided to detect PCR amplification products at 62 ° C. for both probes.
[0223]
2) Probe specificity
FIG. 4 shows the results of real-time monitoring when PCR of the target nucleic acid was performed using the above-mentioned a and b as a nucleic acid probe and primer under the PCR reaction conditions described in 7) of A) above. Indicated. Similarly, FIG. 5 shows the results of real-time monitoring when PCR of the internal standard nucleic acid was performed using the above c and d using NECMB-24 as a nucleic acid probe and primer. It was found that both probes showed a high fluorescence intensity change rate (fluorescence quenching rate) of about 30%. Further, in both probes, the obtained fluorescence intensity change rate changed according to the mixing ratio of the target nucleic acid and the internal standard nucleic acid. From this result, it was considered that the target nucleic acid can be quantitatively quantified by this method.
[0224]
3) Creation of relational expression between DNA concentration and fluorescence intensity change rate
The fluorescence intensity change rate at various DNA concentrations was measured with both probes. The results are shown in FIGS. 6A and 6B (in preparing this figure, the processing described in the data analysis method of the present invention was performed to determine the rate of change in fluorescence intensity).
[0225]
A method for obtaining the concentration or copy number of the target nucleic acid in the unknown sample from each figure is described below. First, an internal standard nucleic acid whose copy number is known, a target nucleic acid probe, and an internal standard nucleic acid probe are added to an unknown sample containing the target nucleic acid, and PCR is performed. When amplification of both nucleic acids becomes visible from the change in fluorescence intensity of the reaction system, the rate of change in fluorescence intensity of both nucleic acids is determined. The fluorescence intensity change rate is applied to FIGS. 6A and 6B, and the amplified internal standard nucleic acid amount (referred to as A) and target nucleic acid amount (referred to as B) when the fluorescence change rate is indicated. When the copy number of the internal standard nucleic acid amount before nucleic acid amplification is A ′, the copy number B ′ of the target nucleic acid before amplification is set. B 'can be determined by the following formula:
B '= (B / A) A'
[0226]
4) Quantification of target nucleic acid by smart cycler using conventional calibration curve
A nucleic acid extraction sample actually extracted from a soil sample was mixed with a target gene sample of a known concentration at different concentrations to examine whether or not accurate quantification could be performed. The internal standard nucleic acid probe and the target nucleic acid probe were both added to the reaction tube, and the internal standard nucleic acid and the target nucleic acid were detected simultaneously.
[0227]
First, a calibration curve was prepared using an external standard gene (nec1 gene) at a known concentration (FIGS. 7A and 7B). The sample containing nucleic acid extracted from soil, 0 μl, 1 μl, 2 μl, 3 μl, with 6,690,000 copies of the target gene (nec1 gene) added as a template, the nec1 gene in this sample was quantified as described above. This was performed using a calibration curve. As a result, it became clear that the graph shifted to the right, that is, the amplification was delayed as the amount of the soil sample to be added increased (FIG. 8). It was found that the Ct value increased due to this delay, and as a result, the quantitative value was calculated lower than the actually added target gene (nec1 gene) (see Table 3). From the above results, it was clarified that the existing method using a calibration curve with a conventional external standard gene (real-time quantitative PCR method) cannot accurately quantify the target gene for samples containing PCR inhibitors. .
[0228]

[0229]
5) Quantification of target nucleic acid by smart cycler using the method of the present invention
The number of copies of the target nucleic acid before amplification was determined by the method of adding the internal standard nucleic acid of the present invention. Table 3 shows differences in quantitative values due to differences in methods. As a result, the graph shifts to the right as the amount of soil sample to be added is the same as described above. In the present invention, however, a decrease in amplification efficiency (amplification delay) was observed. Since gene amplification was similarly reduced, it was clarified that the amount of the added target gene almost coincided with the quantitative value obtained by the method of the present invention.
[0230]
From the above results, the usefulness of the novel measurement method of the present invention for determining the concentration or copy number of the target nucleic acid from the amount or rate of change in fluorescence intensity derived from the target nucleic acid and the internal standard nucleic acid is clarified by adding the internal standard nucleic acid. Became. In addition, the standard deviation value of the method of the present invention was smaller than that of the conventional real-time quantitative PCR method, and it was revealed that the fluctuation of the quantitative value was small. From these results, the superiority of the present invention over the conventional method was proved in the accuracy even when the PCR inhibitor was present as well as when it was not present.
[0231]
6) Implementation of the method of the present invention in a model system
Based on the experimental example, the addition ratio of the target nucleic acid and the internal standard nucleic acid was changed, and the amount of change in fluorescence intensity of the two probes was monitored in real time for each PCR amplification product. As a result, it was confirmed that the amount of change in fluorescence intensity was obtained according to the addition ratio.
[0232]
7) Quantification of actual soil samples
Based on the experimental example, a soil sample (including the target nucleic acid) in which the copy number of the target gene (nec1 gene) is unknown was quantified using a known amount of the internal standard nucleic acid.
About 66.9 copies of the internal standard nucleic acid was added to 1 μl and 2 μl of the soil extracted nucleic acid samples, and the target gene (nec1 gene) was quantified by the method of the present invention. As a result, in the system to which 1 μl of the soil extracted nucleic acid sample was added, it was quantified as 67.5 copies. Further, the quantitative value of the system to which 2 μl of the soil extracted nucleic acid sample was added was 142.5 copies. Since this quantitative value is about twice that of the system to which 1 μl of the soil extracted nucleic acid sample is added, it is judged that the target gene (nec1 gene) in the soil extracted nucleic acid sample can be quantified almost accurately.
[0233]
Example 2
The same examination as in Example 1 was performed using the probe of Mergney et al.
1) Probe synthesis
For the probe synthesis, those synthesized by Japan Genetic Research Institute (http://www.ngrl.co.jp/) were used.
2) Internal standard nucleic acid (DNA gene)
The same as in the first embodiment.
[0234]
3) Preparation of target nucleic acid probe and internal standard nucleic acid probe
The nucleotide sequence of NECB-24 acceptor shown in Table 4 is the nucleotide sequence of the target nucleic acid probe described in Example 1, and the nucleotide sequence of NECMB-24 acceptor is the internal standard nucleic acid probe described in Example 1 as described above. belongs to. In this example, the target nucleic acid probe is called NECB-24 acceptor, and the internal standard nucleic acid probe is called NECMB-24 acceptor. NECB-24 acceptor has 5 ′ end labeled with a fluorescent dye called LCRed640, and NECMB-24 acceptor has 5 ′ end labeled with a fluorescent dye called LCRed705. Both phosphorylated the 3'OH group at the 3 'end. The fluorescent dyes (LCRed705, LCRed640) modified to the above two types of probes function as acceptor dyes for the FRET phenomenon. A probe modified with a donor dye necessary for exciting these dyes is Nec-donor (see Table 4 for the sequence). The 3 ′ end of the probe is modified with FITC, and when each target gene is present, the probe is designed to hybridize with an interval of 2 bases so that the dye-labeled sites face each other (see FIG. 9). Thus, when Nec-donor and NECB-24 acceptor or Nec-donor and NECMB-24 acceptor are hybridized adjacent to each other, the FRET phenomenon occurs and the fluorescent character changes greatly. From the amount of change, the corresponding nucleic acid of each probe set can be detected.
Further, the base sequence of the probe is the same as that of NECMB-24 used in Example 1 in the NECMB-24 acceptor, and the same as that of NECB-24 used in Example 1 in the NECB-24 acceptor. .
[0235]

[0236]
4) PCR was performed under the same conditions as in Example 1 except for the conditions described below.・ Each probe (NECB-24 acceptor, NECBM-24 acceptor, Nec-donor) addition concentration: 200nM
Measuring device: Light cycler system (Roche Corporation) (hereinafter referred to as light cycler for convenience).
・ Fluorescence measurement channel used
Channel 2 (Ex, 470-490 nm; Em, 640 nm).
Channel 3 (Ex, 470-490 nm; Em, 710 nm).
LCRed640 was measured on channel 2 and LCRed705 was measured on channel 3 at the same time.
5) Extraction of DNA from soil
The same method as in Example 1 was performed.
[0237]
B) Experimental example
1) Evaluation of fabricated probe
Acceptor when hybridized with the target nucleic acid probe (Nec-donor and NECB-24 acceptor set) and the corresponding nucleic acid (target nucleic acid or internal standard nucleic acid) of the internal standard nucleic acid probe (Nec-donor and NECMB-24 acceptor set) The prepared probe was evaluated by creating a fluorescence change rate and a dissociation curve. The same reaction buffer as that used in the PCR reaction was used.
[0238]
The results are shown in FIGS. In both probes, the acceptor fluorescence intensity increase rate was about 40%, and the acceptor fluorescence intensity increased remarkably. Similar to NECB-24 and NECMB-24 used in Example 1, Tm values of completely complementary nucleic acids (target nucleic acid and target nucleic acid probe, internal standard nucleic acid and internal standard nucleic acid probe) and mismatched nucleic acid The difference is a little less than 20 ° C., and if it is detected at around 62 ° C., a nucleic acid having a mismatch (a target nucleic acid and an internal standard nucleic acid probe, or an internal standard nucleic acid and a target nucleic acid probe) is detected without being completely complementary. It became clear that only can be detected. From the above results, it was decided that the detection of PCR amplification products was performed at 62 ° C. for both probes, as in Example 1.
[0239]
2) Creation of a relational expression between the increase rate of the fluorescence intensity derived from the target nucleic acid and the increase rate of the fluorescence intensity derived from the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid
Since the target nucleic acid (base sequence and concentration to be added is known, it should be referred to as a standard nucleic acid, but in the present invention, it is referred to as a target nucleic acid for convenience) and an internal standard nucleic acid (with various concentration ratios). PCR was carried out under the reaction conditions described in 7) of A) of Example 1 using the changed product as a template, and the amplification product derived from the target nucleic acid and the amplification product derived from the internal standard nucleic acid were respectively Real-time monitoring was performed with the probe of Mergney et al. From the results, the fluorescence intensity increase rate derived from the amplification product of the target nucleic acid (NECB-24 acceptor probe fluorescence intensity increase rate) and the fluorescence intensity increase rate derived from the internal standard nucleic acid amplification product (NECMB-24 acceptor probe fluorescence intensity increase) The ratio between the target nucleic acid before PCR and the concentration ratio of the internal standard nucleic acid was determined (see FIG. 12). As a result, there is a high correlation between the concentration ratio between the target nucleic acid and the internal standard nucleic acid before PCR and the ratio of the increase in the fluorescence intensity derived from the amplification product of the target nucleic acid and the increase in the fluorescence intensity derived from the amplification product of the internal standard nucleic acid. It became clear that there was. Therefore, it was shown that the concentration ratio between the target nucleic acid before PCR and the internal standard nucleic acid can be known from this relational expression. In preparing this figure, the processing described in the data analysis method of the present invention was performed, and the fluorescence intensity change rate was obtained.
[0240]
A method for obtaining the concentration or copy number of the target nucleic acid in the unknown sample from FIG. 12 will be described below. First, an internal standard nucleic acid whose copy number is known, a target nucleic acid probe, and an internal standard nucleic acid probe are added to an unknown sample containing the target nucleic acid, and PCR is performed. When amplification of both nucleic acids becomes visible from the change in fluorescence intensity of the reaction system, the rate of increase in fluorescence intensity derived from the amplification products of both nucleic acids is determined. From the fluorescence intensity increase rate, the fluorescence intensity increase rate derived from the amplification product of the target nucleic acid and the fluorescence intensity increase rate derived from the amplification product of the internal standard nucleic acid are obtained. By applying this ratio to FIG. 12, the ratio (B / A) between the target nucleic acid (B) and the internal standard nucleic acid (A) is determined. Since the copy number (A ′) of the internal standard nucleic acid before nucleic acid amplification is known, the copy number (B ′) of the target nucleic acid before amplification can be determined by the following formula:
B '= (B / A) A'
[0241]
3) Quantification of target nucleic acid by light cycler using calibration curve of external target (standard) nucleic acid (gene) by conventional method
A nucleic acid extraction sample actually extracted from a soil sample (hereinafter referred to as a soil extraction nucleic acid extraction sample) to a target gene sample having a known concentration under the same conditions as in Example 1 and under the same conditions except that the probe of Mergney et al. Was used. In some cases, it was investigated whether the mixture could be mixed at different concentrations for accurate quantification. It should be noted that the soil to be used was used after confirming that it does not contain the Nec1 gene, which is the target gene. In addition, the internal standard nucleic acid detection probe (NECMB-24 acceptor), the target nucleic acid probe (NECB-24 acceptor), and Nec-donor are both added to the reaction tube to detect the internal standard nucleic acid and the target nucleic acid simultaneously. did.
[0242]
First, 6.690,000 copies of Nec1 gene were added to 3 μl of a sample containing nucleic acid extracted from soil, and nec1 gene was quantified. In addition, the nec1 gene was also quantified in the sample to which the same number of nec1 gene was added without adding the soil-extracted nucleic acid sample. As a result, as in Example 1, it was clarified that the sample to which the soil-extracted nucleic acid sample was added was delayed in comparison with the sample to which the soil-extracted nucleic acid sample was not added. From this result, it was suggested that the soil-extracted nucleic acid sample contains a PCR inhibitor that reduces the amplification efficiency. It was found that due to this delay, the Ct value increased, and as a result, the quantitative value was calculated low (see Table 5). From the above results, it is clear that the conventional method using a calibration curve of external target nucleic acid (gene) (real-time quantitative PCR method) cannot accurately quantify the target gene for samples containing PCR inhibitors. It was.
[0243]
4) Quantification of target nucleic acid by light cycler using the method of the present invention
The method of adding the internal standard nucleic acid of the present invention was performed using the probe of Mergney et al., And the copy number before amplification of the target nucleic acid was determined. Table 5 shows the difference in measured values due to the difference in method. As a result, in the sample to which the soil-extracted nucleic acid sample was added, a decrease in amplification efficiency (a delay in the amplification reaction) was observed as in the conventional method (real-time quantitative PCR method), but the amplification efficiency of the internal standard gene was also increased. Similarly, since it decreased, it became clear that the measured value became constant. Based on this result, the novel measurement method of the present invention, in which an internal standard nucleic acid is added and the concentration or copy number of the target nucleic acid is determined from the amount or rate of change in fluorescence intensity derived from the target nucleic acid and the internal standard nucleic acid, is the probe of Mergney et al. It became clear that it was effective even when using.
In addition, the numerical value of each method of Table 5 is the average value of the numerical value obtained by performing the frequency | count of analysis 5 times.
[0244]

[0245]
5) Measurements on actual soil samples
Based on the above experimental example, the copy number of the soil sample whose copy number of the target gene (nec1 gene) was unknown was measured using a known amount of the internal standard nucleic acid. The used soil sample is the same as that used in the measurement of the actual soil sample of Example 1 (sample measured when 67.5 copies of the target gene (nec1 gene) are present per 1 μl of the soil extraction solution). .
[0246]
As in Example 1, 66.9 copies of the internal standard nucleic acid were added to 1 μl and 2 μl of the soil extracted nucleic acid samples, and the target nucleic acid (gene) (nec1 gene) was measured by the method of the present invention. . As a result, in the system to which 1 μl of the soil extraction nucleic acid sample was added, it was measured to be 83.2 copies. The measurement value was almost the same as the measurement value of Example 1 (67.5 copies). Moreover, the measured value of the system to which 2 μl of the soil extracted nucleic acid sample was added was 152.7 copies. Since the measured value is about twice that of the system to which 1 μl of the soil extracted nucleic acid sample is added, it is judged that the target gene (nec1 gene) in the soil extracted nucleic acid sample can be measured almost accurately. From the above results, it was strongly suggested that the target gene in soil can be accurately measured using the probe of Mergney et al.
[0247]
Example 3
Using the molecular beacon probe (described in (3) of the above-described known invention method), the same examination as in Examples 1 and 2 was performed.
1) Probe synthesis
A probe synthesized by Espec Oligo Service Co., Ltd. (http://www.business-zone.com/espec-oligo/) was used.
[0248]
2) Target nucleic acid probe and internal standard nucleic acid probe
The base sequence of NECB-24 beacon shown in Table 6 is the base sequence of the target nucleic acid probe, and the base sequence of NECMB-24 beacon is that of the internal standard nucleic acid probe. NECB-24 beacon has 4 bases added to form a stem-loop structure. The 5 'end is labeled with a fluorescent dye called FAM and the 3' end is labeled with a quencher substance called DABCYL. ing. NECMB-24 beacon also has 4 bases added to form a stem-loop structure. The 5 'end is a fluorescent dye called 6-TAMRA and the 3' end is a quencher substance called DABCYL. It is labeled.
The probe sequences are as shown in Table 6.
[0249]

[0250]
3) PCR was performed under the same conditions as in Example 2 except for the conditions described below.
・ Each probe concentration: 200nM
Measurement device: Smart cycler system (Takara Bio Inc.) (hereinafter referred to as smart cycler for convenience).
・ Channels used:
Channel 1 (Ex, 450-495 nm; Em, 505-537 nm).
Channel 3 (Ex, 527-555 nm; Em, 565-605 nm).
The fluorescence measurement of FAM labeled with NECB-24 beacon was performed in channel 1 and the fluorescence measurement of 6-TAMRA labeled with NECB-24 beacon was performed simultaneously in channel 3.
[0251]
4) Extraction of DNA from soil
The same method as in Example 1 was performed.
[0252]
B) Experimental example
1) Evaluation of fabricated probe
A fluorescence intensity increase rate and a dissociation curve were prepared when the target nucleic acid probe (NECB-24 beacon) and the corresponding nucleic acid (target nucleic acid or internal standard nucleic acid) of the internal standard nucleic acid probe (NECMB-24 beacon) were hybridized. The same reaction buffer as that used in the PCR reaction was used as described above.
[0253]
The results are shown in FIGS. When fully complementary nucleic acid was added, the fluorescence intensity of both probes increased by about 2,000%. In addition, when a nucleic acid having a mismatch was added, both probes showed almost no increase in fluorescence intensity. From this result, it was determined that both probes hardly hybridize with mismatched nucleic acids (the target nucleic acid and the internal standard nucleic acid probe, and the internal standard nucleic acid and the target nucleic acid probe do not hybridize). From the above results, it was decided that the detection of PCR amplification products was performed at 62 ° C. for both probes as in Example 1.
[0254]
2) Creation of a relational expression between the increase rate of the fluorescence intensity derived from the target nucleic acid and the increase rate of the fluorescence intensity derived from the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid
A relational expression between the increase rate of the fluorescence intensity derived from the target nucleic acid and the increase rate of the fluorescence intensity derived from the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid was determined in the same manner as in Example 2. The method for obtaining the concentration or copy number of the target nucleic acid in the unknown sample is the same as in Example 2.
[0255]
3) Comparison of the target nucleic acid measurement result by the conventional method using the calibration curve of the external standard gene when using Molecular beacon as a probe and the target nucleic acid measurement result by the method of the present invention
Measurement was performed in the same manner as in Examples 1 and 2 except that the molecular beacon probe was used. The measurement results of the target nucleic acid by the conventional method using the calibration curve of the external standard gene were compared with the measurement results of the target nucleic acid by the method of the present invention under the same conditions as the sample. As a result, in the test sample to which the soil-extracted nucleic acid sample was added, a decrease in amplification efficiency (a delay in the amplification reaction) was observed as in Examples 1 and 2, but the added target nucleic acid (gene) was accurately measured. It was possible (see Table 7). From this result, it has been clarified that the novel measurement method of the present invention can be suitably implemented even when molecular beacon is used as a probe.
[0256]

[0257]
Example 4
This is an example to which a method using a nucleic acid probe having the characteristics (1) to (3) of i) to iv) of the method B of the present invention is applied.
1) Target nucleic acid and internal standard nucleic acid
Same as Example 1.
2) Synthesis of target nucleic acid probe
The base sequence was the same as that of NECB-24 described in Example 1 (see Table 2). Then, the fluorescent substance Texas Red was labeled on the 5 'end cytidyl acid, and the quencher substance Dabcyl was labeled in the chain on the 6th thymine from the 5' end.
The synthesis of the nucleic acid probe was outsourced to Espec Oligo Service.
[0258]
3) Synthesis of internal standard nucleic acid probe
The base sequence was the same as that of NECMB-24 described in Example 1, and the fluorescent substance Alexa 594 was labeled in the 5 'terminal cytidylic acid, and the quencher substance Dabcyl was labeled in the chain at the 6th thymine from the 5' end. The preparation was commissioned and synthesized in the same manner as the target nucleic acid probe.
[0259]
4) Hybridization conditions for target nucleic acid and target nucleic acid probe, and internal standard nucleic acid and internal standard nucleic acid probe:
Same as Example 1.
5) Measurement conditions
The excitation wavelength and the measured fluorescence wavelength are as follows.
Target nucleic acid probe: excitation wavelength: 590 nm (4 nm width); measurement fluorescence wavelength: 610 nm (4 nm width).
Internal standard nucleic acid probe: excitation wavelength: 560 nm (4 nm width); measurement fluorescence wavelength: 580 nm (4 nm width).
[0260]
6) Measurement of target gene (nec1 gene) in actual soil nucleic acid extraction sample
The target gene (nec1 gene) in the soil nucleic acid extraction sample was used in the same manner as in Example 1 by using a nucleic acid probe having the characteristics (1) to (3) of i) to iv) of the method B of the present invention. The gene measurement method via the PCR method in which the internal standard nucleic acid was added was measured. PCR conditions and the like are the same as in Example 1. As a result, the target gene (nec1 gene) contained in 1 μL of the soil nucleic acid extraction sample was 63.2 copies, and the target gene (nec1 gene) contained in 2 μL of the soil nucleic acid extraction sample was 148.1 copies. Since this measured value is almost the same as the result obtained in Examples 1 to 3, a nucleic acid probe having the characteristics (1) to (3) of i) to iv) of the method B of the present invention was used. It was also shown that the novel measurement method of the present invention can be suitably implemented.
[0261]
As described above, in Examples 1 to 4, a common target gene (nec1 gene) was measured by the same method (method of adding an internal standard nucleic acid) using different types of nucleic acid probes. As a result, almost the same good results could be obtained. It has been clarified that any probe capable of monitoring the gene amplification process in real time can be applied to the novel measurement method of the present invention regardless of the type of probe.
[0262]
Example 5
Using the hairpin probe described in v) to ix) in the case of utilizing the FRET phenomenon of the known method as a primer (sunrise primer), real-time quantitative PCR is performed to determine the copy number of the target nucleic acid before nucleic acid amplification. It is an Example which applies the method of this invention to the method to obtain | require.
1) Base sequence of target nucleic acid
Necl gene used in Examples 1-3
2) Internal standard nucleobase sequence
Internal standard gene corresponding to Necl gene used in Examples 1-3
[0263]
3) Primer
(1) Forward primer for target nucleic acid detection
・ Base sequence:acagtta CTCCATGAACTGTACCGCGACCAG (underline: added sequence)
-The 5 'end was labeled with the donor dye FAM, and the 12th base T from the 3' end was labeled with the quencher dye DABCYL.
(2) Forward primer for detection of internal standard nucleic acid
・ Base sequence:agctttg CTCCATGAAagcTtCCGCGACCAG (Underline: added sequence, lower case: site where the sequence differs from the forward primer for detecting the target nucleic acid. In other words, the site where the sequence of the target nucleic acid and the internal standard nucleic acid is different)
The phosphorus group at the 5 'end was labeled with the donor dye 6-TAMRA, and T at the 12th base from the 3' end was labeled with the quencher dye DABCYL as described above.
(3) Reverse primer
Base sequence: f (NECS-R) in Table 1 above
[0264]
The forward primer (forward primer for target nucleic acid detection and forward primer for internal standard nucleic acid detection) is designed to function as a sunrise primer, and the intramolecular secondary structure in the primer is eliminated by gene amplification. Thus, it is designed to emit fluorescence. Therefore, the amplification product can be monitored in real time by measuring the fluorescence emission. Each forward primer is designed to recognize different sites in the base sequences of the internal standard gene and the target gene, specifically bind to each other, and extend. Therefore, the target nucleic acid or the internal standard nucleic acid can be specifically amplified by each forward primer (the target primer for detecting the target nucleic acid and the forward primer for detecting the internal standard nucleic acid). In addition, the target nucleic acid detection forward primer and the internal standard nucleic acid detection forward primer are labeled with different fluorescent dyes, so the amplification reaction product derived from the target nucleic acid and the amplification product derived from the internal standard nucleic acid are the same reaction. It can be detected simultaneously in the system.
The primers were synthesized by entrusting to Espec Oligo Service Co., Ltd. (http://www.business-zone.com/espec-oligo/).
[0265]
4) PCR was performed under the following conditions.
-Denaturation reaction: 95 ° C, 15 sec
・ Annealing and detection: 55 ℃, 5sec
・ Extension: 72 ℃, 10sec
-Taq polymerase: Gene Taq (Nippon Gene Co., Ltd.)
・ Primer addition concentration: 100 nM
Template nucleic acid (Template): a mixture of internal standard nucleic acid NECM1 and target nucleic acid NEC1 PCR fragment (a and b plumer as a set, PCR amplification product: about 700 bp)
・ Measuring device: Smart cycler system (Takara Bio Inc.)
[0266]
・ Channels used:
Channel 1 (Ex, 450-495 nm; Em, 505-537 nm).
Channel 3 (Ex, 527-555 nm; Em, 565-605 nm).
FAM labeled with a target primer for detecting a target nucleic acid was measured in channel 1, and at the same time, fluorescence of 6-TAMRA labeled with a forward primer for detecting an internal standard nucleic acid was measured in channel 3.
[0267]
5) Creation of a relational expression between the increase rate of fluorescence intensity derived from the target nucleic acid and the increase rate of fluorescence intensity derived from the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid
PCR was carried out under the reaction conditions described in 4) of Example 2 using variously varied concentration ratios of the target nucleic acid and the internal standard nucleic acid as a template, and the amplification product derived from the target nucleic acid and the internal standard Each amplification product derived from nucleic acid was monitored in real time. From the results, the fluorescence intensity increase rate derived from the amplification product of the target nucleic acid (NECB-24 acceptor probe fluorescence intensity increase rate) and the fluorescence intensity increase rate derived from the internal standard nucleic acid amplification product (NECMB-24 acceptor probe fluorescence intensity increase) The ratio between the target nucleic acid before PCR and the concentration ratio of the internal standard nucleic acid was determined (see FIG. 15). As a result, there is a high correlation between the concentration ratio between the target nucleic acid and the internal standard nucleic acid before PCR and the ratio of the increase in the fluorescence intensity derived from the amplification product of the target nucleic acid and the increase in the fluorescence intensity derived from the amplification product of the internal standard nucleic acid. It became clear that there was. Therefore, it was shown that the concentration ratio between the target nucleic acid before PCR and the internal target nucleic acid can be known from the relational expression. From the above results, in the method using the sunrise primer, as in Examples 2 and 3, the increase rate of the fluorescence intensity derived from the amplification product of the target nucleic acid and the increase in the fluorescence intensity derived from the amplification product of the internal standard nucleic acid. From the ratio to the ratio, it became clear that the concentration ratio between the target nucleic acid and the internal standard nucleic acid before PCR can be determined.
[0268]
4) Measurement of target nucleic acid by smart cycler using calibration curve of conventional method
We examined whether nucleic acid extraction samples that were actually extracted from soil samples were mixed with known concentrations of target gene samples at different concentrations to make accurate measurements. Both the internal standard nucleic acid primer and the target nucleic acid primer were added to the reaction tube, and the internal standard nucleic acid and the target nucleic acid were detected simultaneously.
[0269]
First, a calibration curve with an external target gene (nec1 gene) at a known concentration was prepared. Using a sample obtained by adding 6,690,000 copies of a target gene (nec1 gene) to 3 μl of a sample containing nucleic acid extracted from soil, the nec1 gene in this sample was measured using the above calibration curve. In addition, the nec1 gene was also measured for samples to which the same number of nec1 genes were added without adding the soil-extracted nucleic acid sample. As a result, as in Examples 1 to 3, the addition of the soil-extracted nucleic acid sample caused the graph to shift to the right compared to the sample to which the soil-extracted nucleic acid sample was not added, that is, the amplification reaction was delayed. It has become clear that this happens. Due to this delay, the Ct value increased, and as a result, it was found that the measured value was calculated lower than the actually added target nucleic acid (gene) (nec1 gene) (see Table 8). From the above results, in the conventional method (real-time quantitative PCR method) using a calibration curve with an external target nucleic acid (gene), even when a sunrise primer is used, the target gene in the sample containing the PCR inhibitor is not detected. It became clear that the measurement could not be performed accurately.
[0270]

[0271]
5) Measurement of target nucleic acid by smart cycler using the method of the present invention
The copy number before amplification of the target nucleic acid was determined by the method of adding the internal standard nucleic acid of the present invention (see Table 8). As a result, the graph shifts to the right as the amount of the soil sample to be added is the same as described above, but in the present invention, a decrease in amplification efficiency (a delay in the amplification reaction) was observed. Since the amplification of the internal standard gene was similarly reduced, it was revealed that the amount of the added target gene almost coincided with the measured value by the method of the present invention.
[0272]
Based on the above results, the internal standard nucleic acid corresponding to the target nucleic acid is added to the reaction system, and the amount or rate of change in fluorescence intensity derived from the target nucleic acid and the internal standard nucleic acid is determined using the sunrise primer. The usefulness of the novel measurement method of the present invention for determining the copy number has been clarified.
In addition, when the nucleic acid probe whose fluorescence is reduced by the hybridization described in (ix) of the method B of the present invention is used as a primer, the same tendency as described above is observed. The result shown (data not shown) was obtained. From these results, it was found that any primer capable of monitoring the gene amplification process in real time can be applied to the novel measurement method of the present invention regardless of the type of primer.
[0273]
Example 6
The gene amplification method was changed from the PCR method to the LAMP (Loop-Mediated Isothermal Amplification) method (one nucleic acid amplification method), and the same examination as in Examples 1 to 4 was performed.
1) Target gene
The nec1 gene was used in the same manner as in Examples 1-5.
2) Extraction of DNA from soil
The same method as in Example 1 was performed.
3) Probe synthesis
Probes and primers were consigned to Espec Oligo Service Co., Ltd. (http://www.business-zone.com/espec-oligo/). 4) Preparation of internal standard nucleic acid (DNA gene)
It was prepared by the method for obtaining an artificial gene described later as Example 8.
5) LAMP primer design and sequence
Primer design was performed using primer design software for the LAMP method on the homepage of Eiken Chemical Co., Ltd. (http://www.eiken.co.jp/).
This primer has a sequence completely complementary to the target nucleic acid and the internal standard nucleic acid, and is a primer capable of simultaneously amplifying the target nucleic acid and the internal standard nucleic acid.
[0274]

[0275]
6) Target nucleic acid probe and internal standard nucleic acid probe
NECB-23 LAMP shown in Table 9 is a base sequence of a probe for detecting an amplification product derived from a target nucleic acid amplified by the LAMP method, and NECMB-23 LAMP is derived from an internal standard amplified by LAMP. It is a nucleic acid probe for detecting an amplification product. In this example, the target nucleic acid probe is called NECB-24, and the internal standard nucleic acid probe is called NECMB-24.
[0276]
NECB-24 is labeled with BODIPY FL by the method described above, and NECMB-24 is labeled with 6-TAMRA at the 5 'end by the method described above. Both were used by phosphorylating the 3 'OH group at the 3' end.
This probe is a type of nucleic acid probe used in Example 1 in which the fluorescence intensity decreases by hybridizing with the corresponding nucleic acid.
In the table, the 9, 10, 11, 12, and 14th bases t, a, c, and t of NECB-23 LAMP are changed to A, T, G, and A, respectively, in NECMB-23 LAMP. .
[0277]
B) Experimental example
1) Evaluation of fabricated probe
Fluorescence intensity change rate {quenching (decrease rate) rate} when hybridized with corresponding nucleic acid (target nucleic acid or internal standard nucleic acid) of target nucleic acid probe (NECB-23 LAMP) and internal standard nucleic acid probe (NECMB-23 LAMP) A dissociation curve was generated. The same reaction buffer as that used in the LAMP method was used.
[0278]
The results are shown in FIGS. With NECB-23 LAMP, the difference in Tm between the target nucleic acid and the internal standard nucleic acid is about 20 ° C, and if it is detected in the range of 60 to 70 ° C, only the target nucleic acid is detected without detecting the internal standard nucleic acid. It became clear that it could be detected. Similarly, NECMB-23 LAMP has a difference in Tm value between the internal standard nucleic acid and the target nucleic acid of about 20 ° C. as in the case of NECB-23 LAMP. It was revealed that only the target nucleic acid can be detected well without detecting. Based on the above results, PCR amplification products were detected at 65 ° C., the reaction temperature of the LAMP method, for both probes.
[0279]
2) Gene amplification by the LAMP method was performed under the following conditions.
・ Reaction temperature: 65 ℃, 60min
・ Amplification kit including polymerase, dNTP, etc .: Loopamp DNA amplification reagent kit (Eiken Chemical Co., Ltd.)
・ Primer addition concentration: 800 nM (NEC FIP, NEC BIP), 200 nM (NEC F3, NEC B3) (see Table 10)
・ Probe addition concentration: 200nM each
Template nucleic acid (Template): a mixture of internal standard nucleic acid NECM1 and target nucleic acid NEC1 PCR fragment (amplification product: about 700 bp with a and b primers in Table 1 as a set)
Measurement device: Smart cycler system (Takara Bio Inc.) (hereinafter referred to as smart cycler for convenience).
[0280]
・ Channels used:
Channel 1 (Ex, 450-495 nm; Em, 505-537 nm).
Channel 3 (Ex, 527-555 nm; Em, 565-605 nm).
BODIPY FL was measured on channel 1, and 6-TAMRA fluorescence was measured on channel 3 at the same time.
[0281]

[0282]
3) Creation of a relational expression between the increase rate of the fluorescence intensity derived from the target nucleic acid and the increase rate of the fluorescence intensity derived from the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid
The LAMP reaction was carried out under the reaction conditions described in 2) above using various concentrations of the target nucleic acid and the internal standard nucleic acid as a template, and the amplification product derived from the target nucleic acid and the internal standard nucleic acid were derived. The amplification products were monitored in real time with the nucleic acid probes shown in Table 10, respectively. From the results, the fluorescence intensity change {quenching (decrease) rate} (hereinafter simply referred to as fluorescence quenching rate) derived from the amplification product of the target nucleic acid (NECB-23 LAMP fluorescence quenching rate) and the internal standard nucleic acid amplification product-derived The ratio with the fluorescence quenching rate (the fluorescence quenching rate of NECMB-23 LAMP) was determined, and the relationship between the concentration ratio of the target nucleic acid before the LAMP reaction and the internal standard nucleic acid was determined. As a result, as in the case of PCR shown in Examples 2 to 4, the concentration ratio between the target nucleic acid and the internal standard nucleic acid before amplification, the fluorescence quenching rate derived from the amplification product of the target nucleic acid, and the amplification product derived from the internal standard nucleic acid. A high correlation was found between the ratio to the fluorescence quenching rate (data not shown). Therefore, as in the case of the PCR method, it was shown that the concentration ratio between the target nucleic acid before gene amplification and the internal standard nucleic acid can be known from this relational expression.
[0283]
4) Measurement of target gene (nec1 gene) in soil via LAMP method
Based on the above experimental example, the soil extraction nucleic acid sample (including the target nucleic acid) in which the copy number of the target gene (nec1 gene) is unknown using the internal standard nucleic acid and the novel gene measurement method of the present invention via the LAMP method .) The amount of target nucleic acid present in was measured.
In the experiment, as in the method via PCR in Examples 1 to 5, 66.9 copies of the internal standard nucleic acid were added to 1 μl and 2 μl of the soil-extracted nucleic acid sample, and the target gene (nec1 gene) was measured. The gene measurement method was performed using the LAMP method. The soil extraction nucleic acid sample was the same as in Examples 1-5. As a result, it was measured to be 79.6 copies in the system to which 1 μl of the soil extracted nucleic acid sample was added. Moreover, the measured value of the system to which 2 μl of the soil extracted nucleic acid sample was added was 139.2 copies. Since this measurement value is substantially equivalent to the results obtained in Examples 1 to 5, it is judged that the target gene (nec1 gene) in the soil-extracted nucleic acid sample can be accurately measured.
Based on the above results, the gene amplification method other than the PCR method is added to the novel nucleic acid (gene) measurement method of the present invention, which comprises adding an internal standard nucleic acid and detecting the target nucleic acid and the internal standard nucleic acid with a nucleic acid probe. Proven to be adaptable.
[0284]
Example 7
Streptomyces turgidiscabiesWhen it was necessary to separate and concentrate the nec1 gene from the genomic DNA, it was separated and concentrated by the following method.
The restriction enzymes used were Bfa1, BsaJ1, BssK1, Dde1, Msel, Msp1 (all purchased from New England BIOLABS). Since these are restriction enzymes that recognize and cleave a specific 4-base sequence of double-stranded DNA, the double-stranded DNA is considered to be cleaved to a length of about 40 bases in terms of probability ( Since there are 256 arbitrary 4 base sequences, it is considered that the site cleaved by one type of 4-base recognition restriction enzyme appears every 256 bases. 256 ÷ 6 = 42.6, which is considered to be cleaved about every 40 bases on average). However, when the nec1 gene is treated with the above six restriction enzymes, the nec1 gene has a length of 536 bases and exists as a relatively long double-stranded DNA. For this reason, the fraction containing the target gene can be easily collected using the length of the DNA as an index.
[0285]
1 μl of each of the above six restriction enzymes, 10 μl of x10 buffer attached to Mse1, and a nucleic acid solution (80 μl) extracted in the same manner as in Example 1 from the soil in which potato scab was generated were mixed at 37 ° C. After reacting for 8 hours, it was further reacted at 60 ° C. for 8 hours. nec1 The fraction containing this fragment was passed through a filter {Microcon-100 (divided molecular weight = 100,000), Millipore}. Since only DNA of 100 bp or less passes through this filter, the 536-base nec1 gene remains on the filter. The fraction containing the nec1 gene remaining on the nec1 filter was collected, dried and redissolved in 1 μl of Millipore pure water.
[0286]
The nec1 gene during the re-dissolution was measured by the same procedure as in Experimental Example 7) of Example 1. In order to confirm the presence or absence of concentration, the nec1 gene was also measured in the same manner as described above for the nucleic acid solution before the restriction enzyme treatment. The amount of nucleic acid solution added was 10 μl. As a result, the number of nec1 genes in the nucleic acid solution subjected to the concentration treatment was 4790 copies / μl, and the number of genes in the nucleic acid solution when the concentration was not performed was 63 copies / μl. From the above results, it was proved that the target nucleic acid (nec1 gene) can be easily concentrated by this method.
[0287]
Example 8
Synthesis of a long-chain artificial gene having an arbitrary base sequence was carried out by the following method.
1) Synthesis of single-stranded oligonucleotide DNA
The single-stranded oligonucleotide gene to be synthesized was an internal standard gene corresponding to the nec1 gene used in Example 6. The sequence is shown below. The number of bases is 274 bp, and the part indicated by lowercase letters and underline is a part different in sequence from the target nucleic acid nec1 gene. The single-stranded oligonucleotide DNA was synthesized by entrusting to Espec Oligo Service Co., Ltd.
[0288]
The synthesized oligo DNA was subjected to electrophoresis, and it was confirmed whether a band derived from the desired single-stranded oligo DNA gene (274 bp) was obtained, but no clear band could be confirmed around 270 bp. Moreover, the electrophoretic pattern was smeared as a whole, and no clear band was observed except for 270 bp. The above results are thought to suggest that single-stranded oligo DNAs of various lengths have been synthesized, and the ratio of the desired single-stranded oligo DNA gene is very low. From the above results, it was strongly suggested that it is impossible to obtain a long-chain gene having an arbitrary sequence by a method for synthesizing a normal oligo DNA.
[0289]

[0290]
2) PCR reaction using single-stranded oligonucleotide DNA as a template
In order to obtain the target DNA, PCR amplification was performed using the single-stranded oligonucleotide DNA gene as a template. The primer sequences used are shown in Table 11. PCR conditions are as shown below.
-Denaturation reaction: 95 ° C, 60 sec
・ Annealing and detection: 62 ℃, 60sec
・ Extension: 72 ℃, 60sec
・ Number of cycles: 40 cycles
-Taq polymerase: Gene Taq (Nippon Gene Co., Ltd.)
・ Primer addition concentration: 500nM each
・ PCR device: iCycler (manufactured by Bio-Rad)
[0291]

[0292]
After the PCR reaction, electrophoresis was performed. However, no clear band was confirmed around 270 bp. Therefore, the obtained PCR product was diluted 100-fold, PCR was performed again under the same conditions using this as a template, and the product was confirmed by electrophoresis. As a result, a band was confirmed in the vicinity of 270 bp. However, since the migration pattern was not clear and the whole was smeared, PCR was performed again using a 100-fold diluted PCR product as a template, and when the product was confirmed by electrophoresis, a clear band was found around 270 bp. confirmed. In order to confirm that the target product was actually obtained, the base sequence of the PCR product was determined. The process is shown below.
[0293]
The PCR product was purified using a Microspin S-400HR column (Amersham Pharmacia). The sequencing reaction was carried out using a dideoxy terminator sequencing kit (Applied Biosystems), and the resulting product was obtained using an automatic base sequencer (ABI PRISM TM 377, Applied Biosystems). The nucleotide sequence was determined. As a result, it was confirmed that the PCR product had the sequence of the target internal standard gene.
From the above results, it was proved that a long-chain artificial gene having an arbitrary sequence can be easily synthesized by the method of the present invention.
[0294]
Example 9
The type of dye to be labeled on the nucleic acid probe of the method B of the present invention was examined. A deoxyribopolynucleotide having the following base sequence was used as a nucleic acid probe, and a deoxyribopolynucleotide having the following base sequence was used as a corresponding nucleic acid. Each deoxyribopolynucleotide was prepared using the nucleic acid synthesizer.
[0295]
・ For target nucleic acid probe: 5'CCCCCCCCCTTTTTT3 '
・ For target nucleic acid: 5'AAAAAAGGGGGGGGGGGG3 '
To label the dye on the deoxyribopolynucleotide for the target nucleic acid probe, the same method as in Example 1 was used.
The fluorescence measurement was performed under the following conditions.
[0296]
(1) Components of the hybridization solution
・ Synthetic DNA 320nM (final concentration)
・ Nucleic acid probe 80nM (final concentration)
・ NaCl 50 mM (final concentration)
・ MgCl₂                       1mM (final concentration)
・ Tris-HCl buffer (pH = 7.2) 100 mM (final concentration)
・ Milli Q pure water 1.6992ml
・ Total amount 2.0000ml
(2) Hybridization temperature: 51 ° C
(3) Measurement conditions:
Excitation light: described in Table 9
Measurement fluorescence color: described in Table 9
[0297]
The results are shown in Table 9. As can be seen from the table, preferred examples of the fluorescent dye used for reducing the fluorescence intensity of the fluorescent dye of the present invention include those having a fluorescence intensity decrease rate of 15% or more. .
[0298]
Example 10
Example of applying the method of Morrison et al. (Method using two types of nucleic acid probes. Before the nucleic acid probe hybridizes to the target nucleic acid, the probes are hybridized to each other. ).
1) Target nucleic acid and internal standard nucleic acid
Same as Example 1.
2) Synthesis of target nucleic acid probe
The target nucleic acid probe is composed of the following two oligonucleotides.
a) 5'-TCCATGAACT GTACCGCGAC-CAG-3 '(base sequence obtained by removing the cytidylic acid at the 5' end of the target nucleic acid probe NECB-24 of Example 1)
b) 5'-TCGCGGTACA GTTCATGGA-3 '(base sequence hybridized to a)
In a), the fluorescent substance 6-TAMRA was labeled on the OH group of the phosphate group at the 5 'end.
In b), the quencher dye Dabcyl was labeled on the OH group at the 3'-position C of deoxyribose of adenosine at the 3 'end.
[0299]
3) Internal standard nucleic acid probe
Similar to the target nucleic acid, it is a probe used in the method of Morrison et al.
The following base sequence consists of two oligonucleotides.
a) 5′-TCCATGAAAGCTTCCGCGACCAG-3 ′ (base deleted from 5 ′ end of target nucleic acid probe NECMB-24 of Example 1)
b) 5'-TCGAA GTTCATGGA-3 '(base sequence hybridized to a)
In a), the fluorescent substance FAM was labeled on the OH group of the phosphate group at the 5 'end.
In b), the quencher dye Dabcyl was labeled on the OH group at the 3'-position C of deoxyribose of adenosine at the 3 'end.
[0300]
4) Target nucleic acid probe and internal target nucleic acid probe preparation method
The same operation as in Example 4 was performed.
5) Hybridization conditions for target nucleic acid and target nucleic acid probe, and internal standard nucleic acid and internal standard nucleic acid probe:
Same as Example 4.
[0301]
6) Measurement conditions
The excitation wavelength and the measured fluorescence wavelength are as follows.
Target nucleic acid probe: excitation wavelength: 527 to 555 nm; measurement fluorescence wavelength: 565 to 605 nm.
Internal standard nucleic acid probe: excitation wavelength: 450 to 495 nm; measurement fluorescence wavelength: 505 to 537 nm.
[0302]
7) Measurement of fluorescence intensity of hybridization reaction in a measurement system that does not contain soil sample (inhibitor of hybridization reaction)
Various concentrations of the target nucleic acid and the internal standard nucleic acid were changed, the amount of change in fluorescence intensity before and after hybridization was measured, and a calibration curve for the target nucleic acid and the internal standard nucleic acid was prepared.
Target nucleic acid probe concentration: 500 nM
Concentration of internal standard nucleic acid probe: 500 nM
[0303]
8) Soil sample
A soil containing no target nucleic acid was prepared in the same manner as in Example 1. It was confirmed by the method of Example 1 that the target nucleic acid was not contained.
9) Target nucleic acid recovery test in measurement system containing soil sample (inhibitor of hybridization reaction) Measurement of target nucleic acid
(1) A soil sample was added to 100 μL of the measurement system, and the measurement system was complicated. Furthermore, target nucleic acid is 2.5 × 10⁸A copy was added to make 10 μL. The fluorescence intensity was measured by varying the concentration of the internal standard nucleic acid. As a result, a calibration curve for the internal standard nucleic acid was obtained. The rate of change of the internal standard nucleic acid calibration curve was calculated and applied to the target nucleic acid. As a result, a calibration curve of target nucleic acid in a complex system was obtained (actually, it was performed by the simplified method. The slope (slope) of the two calibration curves for the internal standard nucleic acid was obtained, and the two calibration curves at the same concentration were obtained. The difference was determined, and the slope of the complex calibration curve for the internal standard nucleic acid and the difference between the two calibration curves at the same concentration were applied as is to the target nucleic acid.
[0304]
(2) Copy number of target nucleic acid in soil sample.
The measurement result is 2.8 × 10⁸The copy / 10 μL, and the target nucleic acid recovery rate was 112%.
[0305]
【The invention's effect】
Since the novel nucleic acid measurement method of the present invention is configured as described above, a sample containing a substance that inhibits the hybridization reaction and / or nucleic acid amplification reaction between the target nucleic acid and the target nucleic acid probe, or a polymorphic nucleic acid. In this method, the target nucleic acid can be specifically measured in a trace amount, accurately and simply in a short time. Moreover, a plurality of target nucleic acids can be measured simultaneously.
[Brief description of the drawings]
FIG. 1 shows the position of hybridization in each gene for each primer that hybridizes to the gene (target nucleic acid).
Fig. 2 Dissociation of target nucleic acid (gene (wild type)) or internal standard nucleic acid (nec1 mutated type) and target nucleic acid probe (NECB-24) hybridization product (target nucleic acid probe complex) curve.
Fig. 3 Hybridization product of target nucleic acid (nec1 gene (wild type)) or internal standard nucleic acid (nec1 mutant type gene) and internal standard nucleic acid probe (NECMB-24) (target nucleic acid probe complex) Dissociation curve.
FIG. 4 Real-time monitoring of PCR using a mixture of a target nucleic acid and an internal standard nucleic acid as a template by a target nucleic acid probe.
FIG. 5 shows real-time monitoring of PCR using a mixture of a target nucleic acid and an internal standard nucleic acid as a template using an internal standard nucleic acid probe.
FIG. 6 is a graph showing a relational expression between a DNA concentration and a fluorescence intensity change amount (rate) of a dye labeled on a target nucleic acid probe.
A: Target nucleic acid
B: Internal standard nucleic acid
FIG. 7A: Real-time monitoring of PCR at various DNA concentrations in a measurement system to which no soil sample was added.
B: Calibration curve based on A
FIG. 8 Real-time monitoring by a conventionally known quantitative PCR method. Template nucleic acid: 7 million PCR products that set a and b probes of nec1 gene
[FIG. 9] Positional relationship of probes upon hybridization.
FIG. 10 shows dissociation curves of Nec-donor and NECB-24 acceptor probe sets for the internal standard gene and the nec1 gene.
FIG. 11 shows dissociation curves of Nec-donor and NECMB-24 acceptor probe sets for internal standard gene and nec1 gene.
FIG. 12 is a relational expression between the increase rate of the fluorescence intensity derived from the amplification product of the target nucleic acid and the increase rate of the fluorescence intensity derived from the amplification product of the internal standard nucleic acid with respect to the concentration ratio between the target nucleic acid and the internal standard nucleic acid before amplification.
FIG. 13: Dissociation curves for NECMB-24 beacon internal standard gene and nec1 gene.
FIG. 14: Dissociation curves for NECMB-24 beacon internal standard gene and nec1 gene.
FIG. 15 is a relational expression between the increase rate of the fluorescence intensity derived from the amplification product of the target nucleic acid and the increase rate of the fluorescence intensity derived from the amplification product of the internal standard nucleic acid with respect to the concentration ratio of the target nucleic acid and the internal standard nucleic acid before amplification (sunrise primer If used)
FIG. 16: Dissociation curves for NECB-23 LAMP internal standard gene and nec1 gene.
FIG. 17 shows dissociation curves for NECB-23 LAMP internal standard gene and nec1 gene.

Claims (10)

(I) a target nucleic acid,
(Ii) having a base sequence obtained by substituting a part of the base sequence of the target nucleic acid with another base;
A known amount of an internal standard nucleic acid that can be amplified simultaneously with the target nucleic acid using the same primer,
(Iii) a target nucleic acid probe comprising an oligonucleotide having a base sequence complementary to the target nucleic acid and labeled with at least one fluorescent dye , wherein the Tm value of the complex of the target nucleic acid probe and the target nucleic acid is A target having a difference of 6 ° C. or more compared to the Tm value of the complex of the target nucleic acid probe and the internal standard nucleic acid , and (iv) an oligonucleotide having a base sequence complementary to the internal standard nucleic acid, In addition, it is an internal standard nucleic acid probe that has a base sequence that does not hybridize with the target nucleic acid and is labeled with at least one fluorescent dye that has a fluorescent color different from the fluorescent dye that labels the target nucleic acid probe . Thus, the Tm value of the complex of the internal standard nucleic acid probe and the target nucleic acid is 6 compared with the Tm value of the complex of the internal standard nucleic acid probe and the internal standard nucleic acid. Those having a difference of more than,
In the reaction system including the nucleic acid amplification reaction, the fluorescence intensity derived from each fluorescent dye labeled on the target nucleic acid probe and the internal standard nucleic acid probe of the reaction system is measured, and the obtained measurement value and the internal standard A method for measuring a nucleic acid, comprising measuring the target nucleic acid and / or the target nucleic acid before the nucleic acid amplification reaction from the amount of nucleic acid added. (I) a target nucleic acid,
(Ii) having a base sequence obtained by substituting a part of the base sequence of the target nucleic acid with another base;
A known amount of an internal standard nucleic acid that can be amplified simultaneously with the target nucleic acid using the same primer,
(Iii) a target nucleic acid probe comprising an oligonucleotide having a base sequence complementary to the target nucleic acid and labeled with at least one fluorescent dye , wherein the Tm value of the complex of the target nucleic acid probe and the target nucleic acid is A target having a difference of 6 ° C. or more compared to the Tm value of the complex of the target nucleic acid probe and the internal standard nucleic acid , and (iv) an oligonucleotide having a base sequence complementary to the internal standard nucleic acid, In addition, it is an internal standard nucleic acid probe that has a base sequence that does not hybridize with the target nucleic acid and is labeled with at least one fluorescent dye that has a fluorescent color different from the fluorescent dye that labels the target nucleic acid probe . Thus, the Tm value of the complex of the internal standard nucleic acid probe and the target nucleic acid is 6 compared with the Tm value of the complex of the internal standard nucleic acid probe and the internal standard nucleic acid. Those having a difference of more than,
In the reaction system comprising: a target nucleic acid and a target nucleic acid probe, and an internal standard nucleic acid and an internal standard probe are hybridized, and before and after the reaction, the target nucleic acid probe and the internal standard nucleic acid probe are labeled with each fluorescent dye. A method for measuring a nucleic acid, which comprises measuring a derived fluorescence intensity and measuring a target nucleic acid from the obtained measured value and the added amount of an internal standard nucleic acid.   The target nucleic acid probe is labeled with a donor dye and an acceptor dye, and the internal standard nucleic acid probe is labeled with a donor dye that is different from the acceptor dye and a donor fluorescent dye that labels the target nucleic acid probe. Item 3. The method according to Item 1 or 2.   The target nucleic acid probe is labeled with a donor dye and a quencher dye, and the internal standard nucleic acid probe is labeled with a quencher dye and a donor dye different from the donor fluorescent dye that labels the target nucleic acid probe. The method according to claim 1 or 2.   The method according to any one of claims 1 to 4, wherein the fluorescent dye labeling sites of the target nucleic acid probe and the internal standard nucleic acid probe are both terminal portions of the probe.   The method according to claim 1, further comprising the step of decomposing the target nucleic acid probe and the internal standard nucleic acid probe by a polymerase in the nucleic acid amplification reaction.   The method according to claim 1, wherein the target nucleic acid probe and the internal standard nucleic acid probe are used as primers for the nucleic acid amplification reaction.   The method according to claim 1, wherein the method for amplifying the nucleic acid is any one of a PCR method, an ICAN method, a LAMP method, a NASBA method, an RCA method, a TAMA method, and an LCR method. The method according to claim 8 , wherein the PCR method is a quantitative PCR method or a real-time quantitative PCR method. The method according to claim 2, wherein the temperature of the hybridization reaction is within a range of Tm value ± 10 ° C. of the complex of the target nucleic acid and the target nucleic acid probe.

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