JP2015503324A - 核酸の単離方法及び装置 - Google Patents
核酸の単離方法及び装置 Download PDFInfo
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
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- 159000000003 magnesium salts Chemical class 0.000 description 1
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- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- JACMPVXHEARCBO-UHFFFAOYSA-N n-pentylpentan-1-amine Chemical compound CCCCCNCCCCC JACMPVXHEARCBO-UHFFFAOYSA-N 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- UYCAUPASBSROMS-AWQJXPNKSA-M sodium;2,2,2-trifluoroacetate Chemical compound [Na+].[O-][13C](=O)[13C](F)(F)F UYCAUPASBSROMS-AWQJXPNKSA-M 0.000 description 1
- XCPXWEJIDZSUMF-UHFFFAOYSA-M sodium;dioctyl phosphate Chemical compound [Na+].CCCCCCCCOP([O-])(=O)OCCCCCCCC XCPXWEJIDZSUMF-UHFFFAOYSA-M 0.000 description 1
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- CEYYIKYYFSTQRU-UHFFFAOYSA-M trimethyl(tetradecyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC[N+](C)(C)C CEYYIKYYFSTQRU-UHFFFAOYSA-M 0.000 description 1
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Abstract
Description
実施例
以下の実施例は、主張される本発明を実施するにあたり、当業者にさらなる指針を与えるために組み入れる。したがって、これらの実施例は、付属の特許請求の範囲で規定される本発明を限定するものではない。
抗凝血剤(ヘパリンナトリウム)、0.25μlのプロテアーゼK(20mg/ml)及び2.5μlの溶液(66.87%の塩酸グアニジニウム及び4%のTriton(商標)X−100)を含むヒト全血(2.5μl)試料を、1.5mlの微小遠心管に添加した。
実施例1で用いたストリップと同様な石英ファイバーフィルターのストリップを、実施例1の場合と同じ形で調製された同じ量の試料溶液と接触させ、実施例1の洗浄溶液と同じ洗浄溶液により実施例1の場合と同じ形で洗浄した。
製品添付文書で推奨される操作プロトコールに従い、QIAamp blood miniキット(QIAGEN)を用いて、ヘパリンナトリウム(抗凝血剤)を含む2.5μlのヒト全血から抽出されたDNA溶液(2.5μl)を、2.5μlの水と混合し、1%のアガロースゲルを有する試料ウェルに入れて、実施例2の場合と同じ形で電気泳動を実施した。
実施例1で用いたストリップと同様な、石英ファイバーフィルターの3つのストリップを、実施例1の場合と同じ形でそれぞれ調製された同じ量の3つの試料溶液にそれぞれ接触させ、実施例1の洗浄溶液と同じ3つの洗浄溶液により実施例1の場合と同じ形でそれぞれ洗浄した。
比較例2
製品添付文書で推奨される操作プロトコールに従い、QIAamp blood miniキット(QIAGEN)を用いて、各々が、抗凝血剤(ヘパリンナトリウム)を含む2.5μlのヒト全血から抽出された、2.5μlのDNA溶液の3つの試料の各々を、2.5μlの水と混合し、各々を、実施例1で用いたストリップと同じ石英ファイバーフィルターストリップ上に滴下させた。乾燥後、試料受容部において、Harris Uni−core 1.2mmパンチャーにより、各ストリップから直径1.2mmの小片をパンチした。各小片を、ベータ−アクチンに対するTaqmanリアルタイムPCR用の25μlのマスターミックス(Ex Taq HS、300nMの各ベータ−アクチンプライマー及び100nMのベータ−アクチンプローブを含有する1倍濃度のPCR緩衝液)を含有する25μlのPCRチューブに入れた。
実施例1の場合と同じ形で調製された同じ量の試料溶液を、実施例1で用いたストリップと同じストリップの試料受容部と接触させた。約5秒後に、試料溶液が、ストリップに吸収され、試料受容部の色が、褐色に変わったことが観察された。
比較例3:乾燥なしの単離
実施例1で用いたストリップと同じ石英ファイバーフィルターのストリップを、実施例1の場合と同じ形で調製された同じ量の試料溶液と接触させ、実施例1の洗浄溶液と同じ洗浄溶液により実施例1の場合と同じ形で洗浄した。
図3に示される通りに構成された装置を用いた。実施例1の試料溶液と同様の、調製された試料溶液を、プラスチック筐体の試料用開口部を介して滴下させて、実施例1で用いたストリップと同じ石英ファイバーフィルターストリップの試料受容部と接触させた。
実施例1の場合と同じ形で調製された同じ量の試料溶液を、実施例1のストリップと同じストリップに添加した。乾燥後、直径1.2mmの小片をパンチし、実施例4の場合と同じ形で増幅した。
ヘパリンナトリウム(抗凝血剤)を含むヒト全血(2.5μl)を、2.5μlの水と混合し、実施例1のストリップと同じストリップの試料受容部に滴下した。乾燥後、試料受容部を中心とする直径1.2mmの小片をパンチし、実施例4の場合と同じ形で増幅した。
試料溶液は、ヘパリンナトリウム(抗凝血剤)を含む2.5μlのヒト全血、0.25μlのプロテアーゼK(20mg/ml)、及び5μlの溶液(66.87%の塩化グアニジニウム及び4%のTriton(商標)X−100)を用いて、実施例1の場合と同じ形で調製した。試料溶液を、実施例1で用いたストリップと同じストリップに添加した。試料溶液が収着されたのを観察した後で、実施例1で用いた洗浄溶液と同じ洗浄溶液400μlを洗浄部に添加して、試料受容部を5分間にわたり洗浄した。
Claims (20)
- 核酸を含む試料溶液を試料受容部と接触させることによって、試料溶液を石英ファイバーフィルターの試料受容部に収着させる段階と、
試料受容部から離れる方向に向かうウィッキング力の下で洗浄溶液を試料受容部に流すことによって、核酸の大半を試料受容部の近傍に維持しながら、試料受容部を洗浄段階と
を含む方法。 - 試料溶液がカオトロピック塩を含む、請求項1記載の方法。
- 洗浄溶液がエタノール及びイソプロパノールの1種以上を含む、請求項2記載の方法。
- 石英ファイバーフィルターが、粒径2.2μm以上、基本重量85g/m2、厚さ約300μm〜約600μmの範囲の粒子に対して、約98%のの粒子保持効率を有する、請求項1記載の方法。
- 石英ファイバーフィルターが、試料受容部から離れた洗浄部を含み、洗浄溶液が洗浄部に添加される、請求項1記載の方法。
- 石英ファイバーフィルターが、第1の端部、洗浄部、試料受容部、及び第2の端部を含む長尺ストリップであり、洗浄部が、第2の端部より第1の端部の近くに位置し、試料受容部が、洗浄部より第2の端部の近くに位置する、請求項5記載の方法。
- セルロース、シリカマイクロファイバーフィルター、グラスファイバー、及び石英フィルターの1種以上で作製され、洗浄部及び第2の端部の少なくとも一方と隣接しかつ垂直方向に整列した吸収性パッドを用意することをさらに含む、請求項6記載の方法。
- 洗浄部が、第1の端部であり、洗浄段階において、試料受容部を洗浄溶液の表面の外部に位置させながら、洗浄部を洗浄溶液中に浸漬する、請求項6記載の方法。
- セルロース、シリカマイクロファイバーフィルター、グラスファイバー、及び石英フィルターの1種以上で作製され、石英ファイバーフィルターと隣接及び垂直方向に整列した吸収性パッドを用意することをさらに含み、洗浄溶液を試料受容部に添加する、請求項1記載の方法。
- 洗浄後、石英ファイバーフィルターを乾燥させることをさらに含む、請求項1記載の方法。
- 石英ファイバーフィルターを備える装置であって、石英ファイバーフィルターが、
試料溶液と接触させることによって核酸を含む試料溶液を収着させ、試料受容部から離れる方向に向かうウィッキング力の下で洗浄溶液を試料受容部に流した後に、核酸の大半を試料受容部の近傍に維持するための試料受容部
を含んでいる、装置。 - 石英ファイバーフィルターが、粒径2.2μm以上、基本重量85g/m2、厚さ約300μm〜約600μmの範囲の粒子に対して、約98%のの粒子保持効率を有する、請求項11記載の装置。
- 石英ファイバーフィルターが、試料受容部から空間的に離隔した洗浄部を含む、請求項11記載の装置。
- 石英ファイバーフィルターが、第1の端部、洗浄部、試料受容部、及び第2の端部を含む長尺ストリップであり、洗浄部が、第2の端部より第1の端部の近くに位置し、試料受容部が、洗浄部より第2の端部の近くに位置する、請求項13記載の装置。
- セルロース、シリカマイクロファイバーフィルター、グラスファイバー、及び石英フィルターの1種以上で作製され、洗浄部及び第2の端部の少なくとも一方と隣接しかつ垂直方向に整列して位置する吸収性パッドをさらに備える、請求項14記載の装置。
- 洗浄部が第1の端部である、請求項14記載の装置。
- セルロース、シリカマイクロファイバーフィルター、グラスファイバー、及び石英フィルターの1種以上で作製され、石英ファイバーフィルターと隣接及び垂直方向に整列した吸収性パッドをさらに備える、請求項11記載の装置。
- 石英ファイバーフィルターをその中に封入する筐体をさらに備える、請求項11記載の装置。
- 筐体が、試料受容部を露出させる試料用開口部を含む、請求項18記載の装置。
- 試料溶液が、カオトロピック塩を含み、洗浄溶液が、エタノール及びイソプロパノールの1種以上を含む、請求項11記載の装置。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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CN201110436344.3A CN103173432B (zh) | 2011-12-22 | 2011-12-22 | 分离核酸的方法及装置 |
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