JP6807403B2 - 核酸ターゲットのバックグラウンド増幅を除去する方法 - Google Patents
核酸ターゲットのバックグラウンド増幅を除去する方法 Download PDFInfo
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- JP6807403B2 JP6807403B2 JP2018561087A JP2018561087A JP6807403B2 JP 6807403 B2 JP6807403 B2 JP 6807403B2 JP 2018561087 A JP2018561087 A JP 2018561087A JP 2018561087 A JP2018561087 A JP 2018561087A JP 6807403 B2 JP6807403 B2 JP 6807403B2
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Description
ここに提案された多重検出アッセイは、同じ酵素の混合物において同時に働く複数の分子のプログラムに依存する。各双安定のノード(システム)は、ターゲットに適した変換されたテンプレートの上流に接続され、リポータープローブの下流に接続されるが、各リポータープローブは異なる発光波長を有する。
修正された配列については、本発明の文脈では、次のシンボルが次のものに関係がある:
「bioteg」:トリエチレングリコール結合したビオチン
「p」:3’リン酸エステル修正;
「*」:ホスホロチオエート骨格修正(エキソヌクレアーゼからの保護);
Cy35:シアニン染料3.5蛍光体
BMN3:BMN3蛍光体
Cy5:シアニン染料5蛍光体
ビオチン:アミノエトキシ−エトキシエタノールを結合したビオチン;
Hex:ヘキサクロロフルオレセイン蛍光体
BHQ1 ブラックホール消光剤1
BHQ2:ブラックホール消光剤2
BMNQ530:BMNQ530消光剤
CBe12PS3:C* G* A* TCCTGAATG−CGATCCTGAATG−Phos(SEQ ID NO:1に対応)
CBe12−1PS3:C* G* A* TCCTGAATG−CGATCCTGAAT−Phos(SEQ ID NO:2に対応)
CBe12−2PS3:C* G* A* TCCTGAATG−CGATCCTGAA−Phos(SEQ ID NO:3に対応)
培養の最初では、入力(Be12=CATTCAGGATCG、SEQ ID NO:4に対応)は、0pM(milliQが代わりに使用された)、8pM、200pM及び1nM濃度でチューブに導入された。
Claims (15)
- 核酸ターゲットの等温増幅においてバックグラウンド増幅を除去する方法であって、
バッファ及び酵素を含む混合物を用意するステップと、
第1、第2及び第3のオリゴヌクレオチドを前記混合物の中に加えるステップとを備え、
前記第1のオリゴヌクレオチドは、5’出力部、及び、3’端部を含む増幅オリゴヌクレオチドであって、ニッキング酵素認識部位を含む部分的な繰り返し構造を含み、
前記第2のオリゴヌクレオチドは、5’端部、及び、3’端部を含む漏出吸収オリゴヌクレオチドであって、前記第1のオリゴヌクレオチドの5’出力部に相補的な配列に結合する結合部を含み、前記第1のオリゴヌクレオチドに沿った重合の生成物を結合し、拡張し、非活性化し、ゆっくりリリースすることができ、それにより、閾値効果を誘発し、
前記第3のオリゴヌクレオチドは、特定のターゲットを変換する特定ターゲット変換オリゴヌクレオチドであり、前記第3のオリゴヌクレオチドの3’側は、重合及びニッキングに際してターゲット配列に結合することができ、前記第3のオリゴヌクレオチドは、前記第2のオリゴヌクレオチドの濃度の制御によって調節された閾値を超えた第1のオリゴヌクレオチドを活性化することができる配列を出力し、
前記第1のオリゴヌクレオチドの5’出力部及び第2の漏出吸収オリゴヌクレオチドの結合部と比較して、前記第1のオリゴヌクレオチドの3’端部は、前記第1のオリゴヌクレオチドによって増幅された配列であって、前記第1のオリゴヌクレオチドの全長に相補的な配列、及び、前記第1のオリゴヌクレオチドの5’出力部に相補的な配列を包含する第1のオリゴヌクレオチドによって増幅された配列、並びに、前記第3のオリゴヌクレオチドによって増幅された配列を包含する配列への低減された親和性を有し、
前記第2のオリゴヌクレオチドの3’端部は、前記第1のオリゴヌクレオチドによって増幅された配列であって、前記第1のオリゴヌクレオチドの全長に相補的な配列、及び、前記第1のオリゴヌクレオチドの5’出力部に相補的な配列を包含する配列と相補的であり、前記第2のオリゴヌクレオチドの5’端部は、非活性化するテールを増幅された配列に付加するためのテンプレートとして役立つ、方法。 - 前記酵素は、ポリメラーゼ、ニッキング酵素及びエキソヌクレアーゼのリストから選択され、前記ポリメラーゼ及びニッキング酵素は等温増幅を進行させることができ、前記エキソヌクレアーゼはシステムの飽和を回避することができる、請求項1に記載の方法。
- 前記酵素と前記オリゴヌクレオチドとの混合物が所定の閾値を超える刺激を受けたときにのみ、信号増幅が開始される、請求項1に記載の方法。
- 前記閾値は第2のオリゴヌクレオチドの濃度の制御によって調節される、請求項1に記載の方法。
- 増幅された配列が高い濃度では前記第1のオリゴヌクレオチドの反応が第2のオリゴヌクレオチドの反応より速いが、増幅された配列が低い濃度では前記第2のオリゴヌクレオチドによる反応が第1のオリゴヌクレオチドの反応より速いように、前記第1及び第2のオリゴヌクレオチドの濃度が選択され、それにより、刺激閾値を超えないときには効果的に増幅を除去する、請求項4に記載の方法。
- ターゲット配列は、バイオマーカーとして使用することができる既知の配列のRNA鎖又はDNA鎖であり、その存在又は濃度は、超高感度で、超特異な方法で検出される、請求項1に記載の方法。
- 複数のターゲット配列は、それらの特異なターゲットを独立して検出し報告することができる直交配列を備えた第1、第2及び第3のオリゴヌクレオチドの複数のセットを使用して、同じ試料内において同時に検出される、請求項6に記載の方法。
- 報告プローブである第4のオリゴヌクレオチドが付加される、請求項1〜7のいずれか1項に記載の方法。
- 前記報告プローブは蛍光プローブである、請求項8に記載の方法。
- 前記報告プローブは、増幅オリゴヌクレオチドによって増幅された信号鎖を検出する、請求項8又は9に記載の方法。
- 前記報告プローブは、蛍光色素及び/又は消光剤によって両方の末端で修正される自己相補形構造鎖である、請求項8〜10のいずれか1項に記載の方法。
- 前記報告プローブは、ニッキング認識部位を含んでいるループを備える、請求項8〜11のいずれか1項に記載の方法。
- 増幅オリゴヌクレオチドは、制限酵素の認識部位を含んでいる、請求項1〜12のいずれか1項に記載の方法。
- 二重鎖の増幅テンプレートの低下速度は、ニッカーゼ/制限酵素の比を変えることによって制御される、請求項13に記載の方法。
- 制限酵素は、エキソヌクレアーゼとともに、付加される、請求項13又は14に記載の方法。
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