JP6861465B2 - 核酸増幅 - Google Patents
核酸増幅 Download PDFInfo
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- JP6861465B2 JP6861465B2 JP2015516255A JP2015516255A JP6861465B2 JP 6861465 B2 JP6861465 B2 JP 6861465B2 JP 2015516255 A JP2015516255 A JP 2015516255A JP 2015516255 A JP2015516255 A JP 2015516255A JP 6861465 B2 JP6861465 B2 JP 6861465B2
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- temperature
- amplification
- chelating agent
- nucleic acid
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Description
この開示は、核酸増幅に関する。
多くの酵素(核酸と相互作用するほとんど全ての酵素及びほとんどのプロテアーゼを包含する)は、二価イオン補因子を必要とする。例えば、核酸増幅反応に関わる酵素は、補因子として二価のマグネシウムイオン(Mg++)をしばしば必要とする。
この開示は、酵素反応の制御方法の開発に、少なくとも一部、基づく。
(a)標的を含む試料並びに(b)結合剤、結合剤によって結合したイオン、バッファー、及びイオンが結合剤によって結合しているときにイオンの存在下で第1の活性を有し、イオンが結合剤から遊離しているときにイオンの存在下で第2の異なる活性を有する少なくとも1つの構成成分を含む増幅試薬を含む試薬を含む混合物を形成するステップと、
第1の温度から第2の温度まで混合物の温度を増加させることによって、増幅試薬の少なくとも1つの構成成分の活性を第1の活性から第2の活性に変化させるのに十分な量のイオンを結合剤から放出するステップと
を含む方法を提供する。
この開示は、反応混合物中の二価イオンを可逆的に結合することによって、二価イオン補因子を必要とする酵素の活性を制御することができるという発見に少なくとも一部基づく。例示的な方法では、反応混合物は、酵素及び試薬混合物を組み合わせることによって調製され、反応混合物は溶液中で可逆的に結合した二価イオンを含む。次に、反応混合物のpHを調整して可逆的に結合した二価イオンを放出し、それによって酵素を活性化させる。
本開示を考慮すると、当業者は、用いる具体的な核酸増幅方法の特性に基づいて、第1の温度、第2の温度、温度感受性バッファーの条件、及びpH感受性キレート化剤を選択することができる。高い反応温度が必要とされる場合は、用いる酵素は好熱種(例えば、テルムス・アカティクス(Thermus aquaticus))から取り出すことができる。
pH依存性キレート化剤EGTAの有り無しで、NEAR増幅をホットスタート条件下で実施した。0又は100コピーの精製されたインフルエンザA型ウイルスRNA、及び150nMのフォワード鋳型、250nMのリバース鋳型、及び200nMの分子ビーコンプローブを用いてアッセイを設定した。鋳型及び分子ビーコンプローブの配列は、以下の通りであった:フォワード鋳型、5’−AGACTCCACACGGAGTCTACTGACAGCCAGACA−3’(配列番号1);リバース鋳型、5’−AGACTCCATATGGAGTCTTGATGGCCATCCGAA’(配列番号2);及び分子ビーコンプローブ、5’−6−Fam−CTGGTAGCCAGGCA GCGACCAG−BHQ1−3’(配列番号3)。以下の条件下で反応を実行した:100mMトリス−Cl(20℃でpH7.9)、15mM Na2SO4、15mM(NH4)2SO4、15mM MgSO4、14mM EGTA、1mM DTT、0.1% Triton X−100、0.3mMの各dNTP、19.2UのBst DNAポリメラーゼ及び15UのNt.BstNBIニッキング酵素。アッセイの構成成分を室温で組み合わせ、約20分の間室温に維持し、その後反応を56℃に置いた。リアルタイム蛍光を用いて反応を10分の間モニターした。EGTA及び100コピーのウイルスRNAを含む反応だけで、増幅を観察した(図1)。
凍結乾燥された構成成分を用いて、ホットスタート条件下でNEAR反応を実施した。凍結乾燥された反応ペレットに、50mMトリス−HCl(20℃でpH7.75)、15mM (NH4)2SO4、15mM MgSO4及び15mM EGTAを含有する50μLの再構成バッファーを加えた。凍結乾燥されたペレットからの構成成分は、再構成後の50μLに、50nMフォワード鋳型、750nMリバース鋳型、300nM分子ビーコンプローブ、50mMトレハロース、225mMマンニトール、50mMトリス−HCl(20℃でpH8.5)、1mM DTT、5mM Na2SO4、0.1% Triton X−100、0.3mMの各dNTP、0.2×SYBR Green I、120UのManta DNAポリメラーゼ及び15UのNt.BstNBIニッキング酵素を含んでいた。鋳型及び分子ビーコンプローブの配列は、以下の通りであった:フォワード鋳型、5’−CGACTCCATATGGA GTCCTCGTCAGACCCAAAA−3’(配列番号4)、リバース鋳型、5’−TGACTCCATATGGAGTCTCATCTTTCCGTCCCC−3’(配列番号5)、及び分子ビーコン、5’−Rox−TCGGGGCAGACCCAAAACCCCGA−BHQ2−3’(配列番号6)。マイコバクテリウム・ボビス(Mycobacterium bovis)BCG(ATCC190115株)からの20又は200コピーのゲノムDNAを用いて増幅を実施した。混合物は、15分の間室温に保持した。室温でのインキュベーションの後、反応を56℃に移行し、リアルタイム蛍光を用いて反応を40分の間モニターした。EGTAが反応に存在したとき、鋳型なしの対照と比較して、20又は200コピーの鋳型DNAを用いて有意な増幅が観察された(図2)。
本発明のいくつかの実施形態が記載されている。それにもかかわらず、本発明の精神及び範囲から逸脱することなく、様々な改変を加えることができることが理解されよう。したがって、他の実施形態は以下の特許請求の範囲内である。
Claims (8)
- (a)ポリヌクレオチド及び増幅試薬混合物を第1の温度で組み合わせて、反応混合物を形成するステップであって、前記第1の温度が20℃〜30℃であり、(i)前記増幅試薬混合物がpH感受性キレート化剤、二価イオン、ニッキングエンドヌクレアーゼ、温度感受性バッファー、及び、DNA又はRNAポリメラーゼを含み、前記キレート化剤が、エチレングリコール−ビス(2−アミノエチルエーテル)四酢酸(EGTA)、EGTA誘導体、エチレンジアミン四酢酸(EDTA)及びEDTA誘導体からなる群から選択されており、前記温度感受性バッファーが、トリシン、グリシンアミド、ビシン、グリシルグリシン、トリス(ヒドロキシメチル)メチル−アミノエタンスルホン酸、N−2−アセトアミド−2−アミノエタンスルホン酸、トリス(ヒドロキシメチル)アミノメタン及びこれらの組み合わせからなる群から選択されており、キレート化剤濃度対二価イオン濃度の比が0.5〜2であり、(ii)前記二価イオンが、ポリヌクレオチドの増幅が進行しないように、第1の温度でキレート化剤と可逆的に結合している、ステップと、
(b)第1の温度から50℃〜60℃である第2の温度まで反応混合物の温度を増加するステップと、を含み、
前記二価イオンが溶液中に存在し、第2の温度で前記キレート化剤と結合していないことにより、前記ポリヌクレオチドの増幅が開始する、方法。 - 前記二価イオンが、マグネシウム、カルシウム、銅、亜鉛、マンガン、鉄、カドミウム及び鉛からなる群から選択される、請求項1に記載の方法。
- 前記温度感受性バッファーがトリス(ヒドロキシメチル)アミノメタンを含む、請求項1に記載の方法。
- 前記反応混合物のpHが、第1の温度から第2の温度に反応混合物の温度を変化させることによって調整される、請求項1に記載の方法。
- 前記温度感受性バッファーが−0.04℃−1〜−0.015℃−1のΔpKaを有する、請求項1に記載の方法。
- 前記増幅試薬混合物が、凍結乾燥形態の1つもしくは複数の構成成分を含み、及び/又は、前記増幅試薬混合物の1つもしくは複数の構成成分が、流体装置、カートリッジ又は側方流動装置での使用に適する容器で提供される、請求項1に記載の方法。
- 前記増幅試薬混合物が逆転写酵素を含む、請求項1に記載の方法。
- 前記キレート化剤が、エチレングリコール−ビス(2−アミノエチルエーテル)四酢酸(EGTA)及びEGTA誘導体からなる群から選択される、請求項1に記載の方法。
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