JP2005538929A - 単一サンプルからの核酸及びタンパク質の単離方法 - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
Description
a)前記サンプルから核酸及びタンパク質を単離する集団を単離する工程、好ましくは1以上の特定集団を単離する工程;
b)固体担体への結合に利用できる遊離形態の核酸及びタンパク質を含むサンプルを得るために当該集団、例えば細胞集団の溶解;
c)サンプルと適当な固体相をDNA、好ましくはゲノムDNAが固体担体に結合するような条件下で接触させる工程;
d)この固体相をサンプルの残留物から分離する工程;
e)前記サンプルの残留物と適当な固体相をRNA、好ましくはmRNAが固体担体に結合するような条件下で接触させる工程;
f)この固体相をサンプルの残留物から分離する工程;
g)前記サンプルの残留物と適当な固体相を特定のタンパク質またはタンパク質の集団が固体担体に結合するような条件下で接触させる工程;
h)この固体相をサンプルの残留物から分離する工程。
(a)核酸成分を結合するのに適した固体担体;及び
(b)タンパク質を結合するのに適した固体担体。
さらに、任意で当該キットはバッファー、塩、ポリマー、酵素等を含む。
100mM Tris−HCl pH 7.5
10mM EDTA
2% SDS
または:
100mM TrisCl pH 7.5
10mM EDTA
5% SDS
10mM NaCl
または:
100mM TrisCl pH 7.5
500mM LiCl
10mM EDTA
1% LiDS
。
実施例1
単一のサンプルからの核酸及びタンパク質の複合単離
実験手順:
2×105培養癌細胞(SW480)を含む細胞ペレットを、ペレット生成を向上させるためのダイナビーズEpithelial Enrich(200μg)及びDNAを結合するダイナビーズM270カルボン酸(300μg)を含む200μlの溶解−結合バッファー(100mM Tris−HCl pH7.5、500mM LiCl、10mM EDTA pH8.0、5mM DTT、1%LiDS)中で溶解した。室温、5分間、ローラー上で混合物を培養することによりDNAを単離した。ビーズ−DNA複合物を溶解物の残りから分離し、500μlの洗浄バッファー(10mM Tris−HCl pH7.5、150mM LiCl、1.0mM EDTA)中で3回洗浄した。その後、80℃、5〜10分間、100μlの溶離バッファー(10mM Tris−HCl pH8.0、0.01% Tween20)中で溶離した。当該DNAは例えば、PCRできる状態である(1μlは十分である)。とりわけ、単離ゲノムDNAから得たPCR生成物のゲル分析を示す図3を参照されたい。
実施例2
IMS(免疫磁気分離)による細胞集団の単離
IMSにより単離されたT細胞の異なる集団中のB細胞の存在/共単離について研究した。CD19、CD3、CD8及びCD4に関して陽性の細胞はIMSにより選択し、gDNAを除去し、mRNAをそれから単離細胞集団から抽出し、cDNAを合成した。それから、各サンプル連続希釈についてCD19プライマーを用いてPCRを行い、40,000〜75細胞を示した。この実験の結果は図5aで見ることができ、CD19 RT−PCRの選択性は第1のゲルパネルにおいて示される。残留ゲルはIMSにおいて低レベルの非特異的結合を示し、T細胞集団に関するCD19 PCRは弱いバンドを示したので、ゆえに非常低レベルの非特異的B細胞結合しか起こらなかった。
実施例3
血液サンプル中への癌細胞の混合及びIMSによる癌細胞の単離
培養大腸癌細胞(SW480)を培養基中に再懸濁し、希釈及び粒度測定器中で計算した。異なる数の癌細胞(1×106〜1細胞)を希釈血液サンプル(1ml全血+1ml DPBS)中に混ぜ、または陽性対照として直接用いた。非混合血液サンプルを完全に同様に処理し、陰性対照として用いた。サンプルは4℃に維持した。
DNA単離
洗浄バッファーを除去し、ロゼット細胞(rosetted cells)を、DNA結合ダイナビーズを含む溶解−結合バッファーを加えることにより溶解した。細胞溶解物を室温、ローラー上で5分間培養した。
残留溶解物を前もって洗浄したダイナビーズオリゴ(dT)25を加えることによりmRNA単離に供し、室温、ローラー上で5分間培養した。
実施例4
陰イオン及び陽イオン交換ビーズを用いたタンパク質単離
癌細胞からのタンパク質断片化を50μl(1.5mg)イオン交換ビーズを用いて各サンプルについて行った。陽イオン交換ビーズを100μl 1M NaCl、20mM クエン酸、pH3.5中で15分間、前処理した。それから、ビーズを100μl洗浄バッファー(50mM NaCl、20mM クエン酸、0.5% Tween 20、pH3.5)中で2回洗浄した。陰イオン交換ビーズを100μl 1M NaCl、20mM Tris−HCl pH10.0中で、15分間、前処理した。該ビーズを続いて100μl洗浄バッファー(50mM NaCl、20mM Tris−HCl、0.5Tween20、pH3.5)中で2回洗浄した。
陰イオン/陽イオン交換(図9a):
細胞ペレットを500μl溶解バッファー(20mM Tris、1% Triton−X−100、150mM NaCl、5mM EDTA、pH8)中で溶解し、ローラー上、10分間、4℃で培養した。溶解物を前処理したイオン交換ビーズを含むチューブに移し、ローラー上、15分間、4℃で培養した。ビーズ−タンパク質複合体を20mM Tris−HCl、50mM NaCl、pH8.0中で3回洗浄した。タンパク質を1M NaCl、20mM クエン酸塩、pH3.5を加えることにより陰イオン交換ビーズから溶離し、ローラー上で15分間培養した。タンパク質は1M NaCl、20mM Tris HCl pH10.0を加えることにより陽イオン交換ビーズから溶離し、ローラー上、15分間培養した。SDS−PAGEにより分析した。
細胞から洗浄バッファーを除去。ロゼット細胞(rosetted cells)を、300μl溶解バッファー(20mM クエン酸、50mM NaCl、20mM Tris、1% Triton−X−100、5mM EDTA、pH3.5)中で溶解した。ローラー上で4℃、5分間培養した。溶解物を前処理した陽イオン交換ビーズを含む新しいチューブに移した。ローラー上で4℃、5分間培養した。該ビーズを300μl洗浄バッファー(20mM クエン酸、50mM NaCl、0.5% Tween 20、pH3.5)中で3回洗浄。
実施例5
減少した数の細胞を単離するための技術の縮小
2000、200及び20の癌細胞を1mlの全血中に混合した。細胞単離を実施例3の記載の通りに行い、(500μgオリゴ(dT)ビーズを用いて)mRNAを得た。前述の実験と同じ量のビーズを用いてオリゴ(dT)ビーズ上でcDNAを合成した。PCRをこれらのサンプルについて、CK19、CD14、CD4及びCD19プライマーを用いて行った。分析をサンプルについて行い、20、2及び0.2細胞由来のmRNAを示した(図10a)。全てのサンプルはCK19に関しては陽性結果を与え、癌細胞の存在を示すことがわかる。CD14及びCD4についての結果は陰性であり、低レベルのCD19が検出され、IMSに関して非常に低レベルの非特異的結合を示した。従って、20細胞は容易に単離でき、この方法により検出できる。
Claims (29)
- 同じサンプルから核酸及びタンパク質を単離する方法であって、前記方法は前記サンプルと固体担体を接触させる工程を含み、ここで同じサンプル中に含まれる核酸及びタンパク質成分は別個の固体担体に結合する、方法。
- DNA及びRNAの両方が同じ固体担体に結合する、請求項1の方法。
- DNA及びRNAが別個の固体担体に結合する、請求項1の方法。
- DNA及びRNAが別個の工程において異なる固体担体に結合する、請求項1の方法。
- RNAとタンパク質、またはDNAとタンパク質、またはDNA、RNAとタンパク質を同じサンプルから単離する、請求項1〜4のいずれかの方法。
- 前記RNAがmRNAである、請求項5の方法。
- 前記サンプルが食品または関連製品であり、または臨床、環境、または生体サンプルである、請求項1〜6のいずれかの方法。
- 前記サンプルと前記固体担体を接触させる前に、該サンプルを核酸及び/またはタンパク質成分をそれらが含まれる構造または構成要素から遊離させるための予備処理工程に供する、請求項1〜7のいずれかの方法。
- 前記サンプルと前記固体担体を接触させる前に、該サンプルを細胞単離手順に供する、請求項1〜8のいずれかの方法。
- 1以上の特定細胞集団を特異的に単離する、請求項9の方法。
- 単離に用いるサンプルまたは細胞集団を、該サンプルと固体担体との接触前に、細胞溶解工程に供する、請求項1〜10のいずれかの方法。
- 前記サンプル内または前記サンプルから単離した細胞の細胞表面タンパク質を細胞溶解工程前に、in vitro修飾手順に供する、請求項11の方法。
- サンプルが前記方法のどの段階においても分割されない、請求項1〜12のいずれかの方法。
- 細胞単離及び/または溶解後、または前記予備処理工程後にサンプルを分割する、請求項1〜12のいずれかの方法。
- 前記サンプルを連続的に、または同時に、または並列に前記固体担体と接触させる、請求項1〜14のいずれかの方法。
- 第1の工程においてDNAを前記サンプルから単離し、第2の工程においてRNAを前記サンプルから単離し、第3の工程においてタンパク質を前記サンプルから単離し、これらの工程を任意の順序で行う、請求項15の方法。
- DNAが表面カルボキシル基を運ぶ担体上で単離される、請求項1〜16のいずれかの方法。
- 合成洗剤の存在下、DNAを固体担体に結合することにより単離する、請求項1〜17のいずれかの方法。
- 細胞溶解及び固体担体への核酸またはDNA結合が同時にまたは付随して起こる、請求項9〜18のいずれかの方法。
- RNAを固体担体により運ばれる、または固体担体に結合した、または結合することができるRNA特異的捕捉プローブを用いて単離する、請求項1〜19のいずれかの方法。
- 前記捕捉プローブがdTオリゴヌクレオチドまたはdUオリゴヌクレオチドである、または含む、請求項20の方法。
- タンパク質を固体担体により運ばれる、または固体担体に結合した、または結合できる適当な結合パートナー/配位子を用いて単離する、請求項1〜21のいずれかの方法。
- タンパク質をクロマトグラフ相互作用を生じることができる表面を有する固体担体を用いて単離する、請求項1〜21のいずれかの方法。
- 前記固体担体が粒子を含む、請求項1〜23のいずれかの方法。
- 前記粒子が磁気粒子である、請求項24の方法。
- 同じサンプルから核酸及びタンパク質を単離するためのキットであり、以下を含む:
(a)核酸成分を結合するのに適した固体担体;
(b)タンパク質を結合するのに適した固体担体。 - (a)の固体担体がDNAまたはRNAに選択的に結合する担体、または両タイプの担体を含む、請求項26のキット。
- キットが(c)特定細胞集団の単離に適した固体担体及び/または(d)前記細胞を溶解する手段、及び/または(e)核酸及び/またはタンパク質を検出する手段も含む、請求項26または27のキット。
- 分析及び/またはmRNA及び/またはタンパク質発現及び/またはゲノム情報とそれらの相互関係の比較のための請求項1〜28のいずれかの方法の使用。
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GB0200927A GB0200927D0 (en) | 2002-01-16 | 2002-01-16 | Method |
GB0227239A GB0227239D0 (en) | 2002-11-21 | 2002-11-21 | DNA/RNA and protection isolation from a single sample |
PCT/GB2003/000156 WO2003062462A2 (en) | 2002-01-16 | 2003-01-16 | Method for isolating nucleic acids and protein from a single sample |
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EP (1) | EP1468116A2 (ja) |
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CN (1) | CN1617938A (ja) |
AU (1) | AU2003202026A1 (ja) |
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- 2003-01-16 WO PCT/GB2003/000156 patent/WO2003062462A2/en active Application Filing
- 2003-01-16 AU AU2003202026A patent/AU2003202026A1/en not_active Abandoned
- 2003-01-16 US US10/501,162 patent/US8110351B2/en not_active Expired - Fee Related
- 2003-01-16 CN CNA038023733A patent/CN1617938A/zh active Pending
- 2003-01-16 JP JP2003562329A patent/JP2005538929A/ja active Pending
- 2003-01-16 CA CA002473376A patent/CA2473376A1/en not_active Abandoned
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2012
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011516064A (ja) * | 2008-04-05 | 2011-05-26 | シングル セル テクノロジー,インコーポレイテッド | 生理活性物質の生産のための単一細胞を選抜する方法 |
JPWO2013145431A1 (ja) * | 2012-03-30 | 2015-12-10 | 株式会社日立製作所 | 微量サンプル由来cDNA増幅法 |
Also Published As
Publication number | Publication date |
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CN1617938A (zh) | 2005-05-18 |
WO2003062462A3 (en) | 2003-12-11 |
CA2473376A1 (en) | 2003-07-31 |
AU2003202026A1 (en) | 2003-09-02 |
US8110351B2 (en) | 2012-02-07 |
US20120178090A1 (en) | 2012-07-12 |
EP1468116A2 (en) | 2004-10-20 |
US20050239068A1 (en) | 2005-10-27 |
WO2003062462A2 (en) | 2003-07-31 |
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