JP2013516185A - 磁性ガラス粒子からの核酸の改善された回収 - Google Patents
磁性ガラス粒子からの核酸の改善された回収 Download PDFInfo
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- JP2013516185A JP2013516185A JP2012547490A JP2012547490A JP2013516185A JP 2013516185 A JP2013516185 A JP 2013516185A JP 2012547490 A JP2012547490 A JP 2012547490A JP 2012547490 A JP2012547490 A JP 2012547490A JP 2013516185 A JP2013516185 A JP 2013516185A
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- nucleic acid
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- ethylene
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- 229940032753 sodium iodate Drugs 0.000 description 1
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- 229910001948 sodium oxide Inorganic materials 0.000 description 1
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- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
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Abstract
Description
核酸の単離は、分子診断における重要なステップである。試料から単離される核酸の質及び量は、下流における診断応用の成功に大きく影響を及ぼす。臨床及びフィールド応用においても、迅速であって且つ自動化になじみやすいことが単離手順に要求される。
本発明は、核酸を含有する試料溶液から核酸を分離する方法であって、以下のステップ:核酸と結合することができる固相、及びエチレン−アミン化合物を含む結合混合物と、前記試料溶液とを接触させ;前記核酸が前記固相と結合し得る条件下、前記固相を含有する前記試料溶液をインキュベートし;そして前記溶液から前記固相を分離することを含む、前記方法である。本発明は場合により、溶出に先立ち、結合した核酸を洗浄するステップを含む。幾つかの実施形態において、本方法は、単離後に核酸を検出することを含む。本発明は、核酸を単離するための反応混合物をさらに含み、前記混合物は、核酸と結合することができる固相、及びエチレン−アミン化合物を含有する。本発明はまた、核酸を試料から分離するためのキットであって:核酸と結合することができる固相、及びエチレン−アミン化合物を含む前記キットを含む。
定義
本明細書において、用語「エチレン−アミン化合物」は、一般式(NH2−CH2=CH2)n−NH2(式中、nは1〜10又はそれ以上である)を有する分子のことである。本発明において有用であるエチレン−アミン化合物は、限定されないが、エチレン−ジアミン(EDA)、ジエチレン−トリアミン(DETA)、トリエチレン−テトラミン(TETA)、テトラエチレン−ペンタミン(TEPA)及びペンタエチレン−ヘキサミン(PEHA)を含む。当業者であれば、他のエチレン−アミン化合物が本発明に有用であると認識し得る。
本発明の範囲を限定するものではなく、説明のみのために、マイコバクテリウム・ツベルクローシス(Mycobacterium tuberculosis)(MTB)からの核酸の単離に本発明が適用された。
溶解のために、100μLのマイコバクテリウム・ツベルクローシス(MTB)細胞懸濁物、又は同量のMTB DNAを含む試料の67 mMホスフェートpH6.8溶液を、400μlの溶解試薬(50mM NaOH、1%Triton X−100、1mM EDTA、0.05%NaN3、pH12+)と混合し、15分間95℃でインキュベートした。その後、400μLの中和試薬(8〜64mMエチレン−アミン、200mM Tris、8〜23mM MgCl2又はMg(OAc)2、0.05%NaN3、pH7.5)を加えた。その後、100μLの磁性ガラス粒子(Roche Molecular Systems,Inc.,Branchburg,N.J.)を加え、そして当該試料を5〜30分間室温若しくは37℃でインキュベートした。磁性ガラス粒子をその後、上清を除去することによって分離し、7.5mMクエン酸ナトリウム二水和物、0.05%(w/v)MIT、pH4.1で2回洗浄した。50〜100μLの溶出試薬(30mM Tris、pH8.5、0.2%w/v 4−ヒドロキシ安息香酸メチル(メチルパラベン)、0.09%NaN3)を用いて核酸を溶出させた。溶解後の手順を、手動で、又はHamilton Star装置(Hamilton Robotics,Inc.,Reno,Nev.)によって自動的に行った。
定義
本明細書において、用語「エチレン−アミン化合物」は、一般式NH 2 −CH 2 −CH 2 −(NH−CH 2 −CH 2 ) n −NH 2 (式中、nは0〜10又はそれ以上である)を有する分子のことである。本発明において有用であるエチレン−アミン化合物は、限定されないが、エチレン−ジアミン(EDA)、ジエチレン−トリアミン(DETA)、トリエチレン−テトラミン(TETA)、テトラエチレン−ペンタミン(TEPA)及びペンタエチレン−ヘキサミン(PEHA)を含む。当業者であれば、他のエチレン−アミン化合物が本発明に有用であると認識し得る。
Claims (19)
- 核酸を含有する試料溶液から核酸を分離する方法であって、以下のステップ:
a.核酸と結合することができる固相、及びエチレン−アミン化合物を含む結合混合物と、前記試料溶液とを接触させ;
b.前記核酸が前記固相と結合し得る条件下、前記固相を含有する前記試料溶液をインキュベートし;そして
c.前記溶液から前記固相を分離すること
を含む、前記方法。 - 前記固相に結合した核酸を洗浄することをさらに含む、請求項1に記載の方法。
- 前記固相から核酸を溶出することをさらに含む、請求項1に記載の方法。
- 前記試料溶液が細胞の溶解によって得られる、請求項1に記載の方法。
- 前記試料溶液が、バチルス属(Bacillus)、クロストリジウム属(Clostridium)、スタヒロコッカス属(Staphylococcus)、ストレプトコッカス属(Streptococcus)、エンテロコッカス属(Enterococcus)、マイコバクテリウム属(Mycobacterium)、及びそれらの組み合わせから成る群から選択されるグラム陽性細菌の溶解によって得られる、請求項4に記載の方法。
- ステップaにおける前記結合混合物が、金属イオンをさらに含む、請求項1に記載の方法。
- ステップaにおける前記結合混合物が、マグネシウムイオン又はマンガンイオンを含む、請求項6に記載の方法。
- ステップaにおける前記結合混合物が、アルカリ金属の水酸化物及び洗剤をさらに含む、請求項1に記載の方法。
- ステップaにおける前記結合混合物が、溶液1(50mM NaOH、1%Triton X−100、1mM EDTA、0.05%NaN3、pH12+)及び溶液2(10〜65mMエチレン−アミン、200mM Tris、5〜25mM MgCl2、0.05%NaN3、pH7.5)の、等しい体積での混合物を含む、請求項8に記載の方法。
- 核酸と結合することができる前記固相が、磁性コア、及びシリカを含む外層を有する粒子を含む、請求項1に記載の方法。
- 固相を使用して試料から核酸を分離するための反応混合物であって:核酸と結合することができる固相、及びエチレン−アミン化合物を含む、前記反応混合物。
- アルカリ金属の水酸化物及び洗剤をさらに含む、請求項11に記載の反応混合物。
- 金属イオンをさらに含む、請求項11に記載の反応混合物。
- マグネシウムイオン又はマンガンイオンを含む、請求項13に記載の反応混合物。
- 核酸と結合することができる前記固相が、磁性コア、及びシリカを含む外層を有する粒子を含む、請求項11に記載の反応混合物。
- 固相を使用して試料から核酸を分離するためのキットであって:核酸と結合することができる固相、及びエチレン−アミン化合物を含む、前記キット。
- 溶解試薬、中和試薬、洗浄試薬及び溶出試薬の1つ又は2つ以上をさらに含む、請求項16に記載のキット。
- 核酸と結合することができる前記固相が、磁性コア、及びシリカを含む外層を有する粒子を含む、請求項16に記載のキット。
- 前記溶解試薬がアルカリ金属の水酸化物及び洗剤を含み、中和試薬がエチレン−アミン化合物、マグネシウムイオン又はマンガンイオン、及び緩衝液を含む、請求項16に記載のキット。
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WO2011083076A1 (en) | 2011-07-14 |
EP2521779A1 (en) | 2012-11-14 |
ES2530466T3 (es) | 2015-03-02 |
EP2521779B1 (en) | 2014-12-10 |
CA2786644C (en) | 2015-08-11 |
US8420801B2 (en) | 2013-04-16 |
JP5715158B2 (ja) | 2015-05-07 |
US20110172408A1 (en) | 2011-07-14 |
WO2011083076A8 (en) | 2014-08-07 |
CN102834518A (zh) | 2012-12-19 |
CN102834518B (zh) | 2015-04-01 |
CA2786644A1 (en) | 2011-07-14 |
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