JP4456046B2 - 磁性色素 - Google Patents
磁性色素 Download PDFInfo
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- JP4456046B2 JP4456046B2 JP2005248331A JP2005248331A JP4456046B2 JP 4456046 B2 JP4456046 B2 JP 4456046B2 JP 2005248331 A JP2005248331 A JP 2005248331A JP 2005248331 A JP2005248331 A JP 2005248331A JP 4456046 B2 JP4456046 B2 JP 4456046B2
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Description
[1] 核酸における酵素反応を行う方法であって、ここで液体中に核酸を含む試料由来の核酸が、ガラス表面を有する磁性粒子への吸着により天然型で単離され、その後酵素反応における基質として使用される、方法;
[2] (a)液体中に核酸を含む試料中の核酸を、ガラス表面を有する磁性粒子に、該核酸が天然型で直接表面に結合できる条件下で吸着させる工程、
(b)結合した核酸を該液体から分離し、その後洗浄溶液を用いて精製する工程、
(c)任意に、乾燥する工程、
(d)溶出バッファーを用いて溶出する工程、および
(e)溶出された核酸を酵素反応における基質として使用する工程
を含む、[1]記載の方法;
[3] 前記核酸がDNAまたはRNAであることを特徴とする、[1]または[2]記載の方法;
[4] 酵素反応のインヒビターが単離の間に取り除かれることを特徴とする、[1]〜[3]いずれか記載の方法;
[5] 長い核酸が単離において分画されないことを特徴とする、[1]〜[4]いずれか記載の方法;
[6] 前記磁性粒子が100μm未満の平均粒子径を有することを特徴とする、[1]〜[5]いずれか記載の方法;
[7] 前記磁性粒子が、その上に外部ガラス表面が適用される、例えば複合材料の内部核または鉄の核を含むことを特徴とする、[1]〜[6]いずれか記載の方法;
[8] 前記核が、酸化鉄が組み込まれる、結晶性のまたはセラミックのまたはガラス様の構造から構成されることを特徴とする、[7]記載の方法;
[9] 前記核がマグネタイト(Fe3O4)またはFe2O3からなることを特徴とする、[7]または[8]記載の方法;
[10] 前記磁性粒子が強磁性であることを特徴とする、[1]〜[9]いずれか記載の方法;
[11] 前記核酸がカオトロピック塩の存在下で単離されることを特徴とする、[1]〜[10]いずれか記載の方法;
[12] カオトロピック塩の濃度が、2〜8mol/l、好ましくは4〜6mol/lであることを特徴とする、[11]記載の方法;
[13] 前記カオトロピック塩が、ヨウ化ナトリウム、過塩素酸ナトリウム、チオシアン酸グアニジウム、イソチオシアン酸グアニジウムおよび塩酸グアニジウムから選択されることを特徴とする、[11]または[12]記載の方法;
[14] 前記試料が前記磁性粒子と混合され、結合のために十分な時間、好ましくは10秒〜30分間インキュベートされることを特徴とする、[1]〜[13]いずれか記載の方法;
[15] 前記核酸がインキュベーション後、磁界の助けを借りて液体から分離されることを特徴とする、[14]記載の方法;
[16] 前記磁性粒子が1回または数回、洗浄溶液を用いて洗浄されることを特徴とする、[1]〜[15]いずれか記載の方法;
[17] 乾燥工程が最後の洗浄工程の後に行われ、任意にアセトンを用いて前処理されることを特徴とする、[16]記載の方法;
[18] 精製された核酸が、好ましくは低い塩容量、特に好ましくは0.2mol/l未満の塩容量の溶出バッファーを使用して、磁性粒子から溶出されることを特徴とする、[16]または[17]記載の方法;
[19] 前記溶出バッファーがTrisを含むか、または脱イオン水であることを特徴とする、[18]記載の方法;
[20] 配列決定、放射性もしくは非放射性標識、1以上の配列の増幅、転写、標識されたプローブ核酸とのハイブリダイゼーション、翻訳、またはライゲーションが酵素反応として行われることを特徴とする、[1]〜[19]いずれか記載の方法;
[21] 単一試験管法として行われることを特徴とする、[1]〜[20]いずれか記載の方法;
[22] 自動化されていることを特徴とする、[1]〜[21]いずれか記載の方法;
[23] 酵素反応における基質としての核酸の使用であって、前記核酸が、ガラス表面を有する磁性粒子への吸着により、天然型で試料から単離されていることを特徴とする、使用;
[24] 前記核酸が、高い塩濃度を有する溶液から低い塩濃度を有する溶液に移されていることを特徴とする、[23]記載の使用;
[25] 前記核酸が濃縮されていることを特徴とする、[23]または[24]記載の使用;
[26] (a)ガラス表面を有する磁性粒子、
(b)試料を溶解させるためのカオトロピック塩溶液、
(c)任意に、洗浄溶液、および
(d)任意に、溶出バッファー
を含む試料から核酸を単離するための試薬組成物;
[27] 前記磁性粒子が100μm未満の平均粒子径を有することを特徴とする、[26]記載の組成物;
[28] 前記磁性粒子が、その上に外部ガラス表面が適用される、例えば複合材料の内部核または鉄の核を含むことを特徴とする、[26]または[27]記載の組成物;
[29] 前記核が、酸化鉄が組み込まれる、結晶性のまたはセラミックのまたはガラス様の構造で構成されることを特徴とする、[28]記載の組成物;
[30] 前記核がマグネタイト(Fe3O4)またはFe2O3からなることを特徴とする、[28]または[29]記載の組成物;
[31] 前記磁性粒子が強磁性であることを特徴とする、[26]〜[30]いずれか記載の組成物;
[32] 前記カオトロピック塩溶液中のカオトロピック塩の濃度が、2〜8mol/l、好ましくは4〜6mol/lであることを特徴とする、[26]〜[31]いずれか記載の組成物;
[33] 前記カオトロピック塩が、ヨウ化ナトリウム、過塩素酸ナトリウム、チオシアン酸グアニジウム、イソチオシアン酸グアニジウムおよび塩酸グアニジウムから選択されることを特徴とする、[26]〜[32]いずれか記載の組成物;
[34] 前記溶出バッファーが低い塩容量、好ましくは0.2mol/l未満の塩容量を有することを特徴とする、[26]〜[33]いずれか記載の組成物;
[35] 前記溶出バッファーがTrisを含むかまたは脱イオン水であることを特徴とする、[26]〜[34]いずれか記載の組成物;
[36] 試料から核酸を天然型で単離するための、[26]〜[35]いずれか記載の試薬組成物の使用;
[37] 前記試料が臨床試料であることを特徴とする、[36]記載の使用;
[38] 前記試料が血液、血清、口腔洗液、尿、脳漿、痰、糞便、生検標本および骨髄試料から選択されることを特徴とする、[36]または[37]記載の使用;
[39] 前記試料が環境分析、食品分析また分子生物学的研究の分野からの試料であり、好ましくは微生物培養物、ファージ溶解物または増幅方法(例えばPCR)の産物由来の試料であることを特徴とする、[36]記載の使用;
[40] 前記核酸が酵素反応のための基質として使用されることを特徴とする、[26]〜[39]いずれか記載の使用;
[41] 前記核酸が、配列決定、放射性もしくは非放射性標識、1以上の配列の増幅、転写、標識プローブ核酸とのハイブリダイゼーション、翻訳またはライゲーションのための基質として使用されることを特徴とする、[36]〜[40]いずれか記載の使用;
[42] 前記核酸が配列決定、放射性もしくは非放射性標識、1以上の配列の増幅、転写、標識プローブ核酸とのハイブリダイゼーション、翻訳またはライゲーションのための基質として使用される、[41]記載の使用;
[43] 実質的に無孔または10nm未満の直径の孔を有するガラスの外表面を有してなり、ここで該ガラスは酸化ホウ素を含有する、磁性粒子;
[44] 粒子が、100μm未満の平均粒径を有することを特徴とする、[43]記載の磁性粒子;
[45] 粒子が、10〜60μmの粒径を有することを特徴とする、[43]または[44]記載の粒子;
[46] 表面に存在し得る孔が、1nm未満の直径を有することを特徴とする、[43]または[44]記載の粒子;
[47] 粒子が、強磁性の核を有することを特徴とする、[43]〜[46]いずれか記載の粒子;
[48] 核が、Fe2O3またはFe3O4を含有することを特徴とする、[47]記載の粒子;
[49] 粒子が、雲母の核および該雲母に固定された磁性粒子を含有する複合物質を含み、該複合物質はガラス層に埋め込まれていることを特徴とする、[43]または[44]記載の粒子;
[50] a) 磁性核を調製する工程、ならびに、
b) (i) ネットワーク形成成分のアルコキシドを含むアルコール溶液から形成されるゾルを表面上に沈積させる工程、
(ii) 噴霧乾燥過程によりゾル層をゲル層に変換させる工程、および
(iii) 続いてゲルを高密度化させる工程
を含む実質的に無孔のガラス表面で磁性核を包み込む工程
により製造され得る、[43]〜[49]いずれか記載の粒子;ならびに
[51] TiO2で被覆された雲母の核および該雲母に固定された磁性粒子からなる複合物質を含み、該複合物質はガラス層に埋め込まれている、[43]記載の粒子
に関する。
B2 O3 (0〜30%)
Al2 O3 (0〜20%)
CaO (0〜20%)
BaO (0〜10%)
K2 O (0〜20%)
N2 O (0〜20%)
MgO (0〜18%)
Pb2 O3 (0〜15%)
などの他の物質を含んでもよい。
図2は、アガロースゲルでの本発明の単離核酸の分離を示したものである。
図3は、本発明による単離およびPCR法による増幅後の反応生成物の分離を示したものである。
図4は、実施例4の結果を生じたゲルを示したものである。
本発明に記載の磁性粒子の製造
6つの異なるゾルを用いた。下記の如く該ゾルを製造した:
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
86.6ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+14.1ml 0.15M HCl
二層混合物を製造する。一層になるまで、二層混合物を室温で攪拌する。
+37.8ml トリメチルホウ酸
を滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+14.1ml 0.15M HCl
を添加する。
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
100.5ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+16.3ml 0.15M HCl
二層混合物を製造した。一層になるまで、二層混合物を室温で攪拌する。
+25.6ml トリメチルホウ酸
を滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+16.3ml 0.15M HCl
を添加する。
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
107.8ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+17.5ml 0.15M HCl
二層混合物を製造した。一層になるまで、二層混合物を室温で攪拌する。
+19.4ml トリメチルホウ酸
を滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+17.5ml 0.15M HCl
を添加する。
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
100.5ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+16.3ml 0.15M HCl
二層混合物を製造した。一層になるまで、二層混合物を室温で攪拌する。
+25.6ml トリメチルホウ酸
滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+16.3ml 0.15M HCl
+1.63g P2 O5
を添加する。
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
100.5ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+16.3ml 0.15M HCl
二層混合物を製造した。一層になるまで、二層混合物を室温で攪拌する。
+25.6ml トリメチルホウ酸
を滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+16.3ml 0.15M HCl
+3.06g Al2 Cl3
を添加する。
250mlの丸底フラスコ中で、常に攪拌しながら、合成を行った。
100.5ml オルトケイ酸テトラエチル
+7ml 無水、非変性エタノール
+16.3ml 0.15M HCl
二層混合物を製造した。一層になるまで、二層混合物を室温で攪拌する。
+25.6ml トリメチルホウ酸
+5.15ml ジルコン(IV)−プロイレート(zircon(IV)−
proylate)、1−プロパノール中70重量%溶液
を滴下にて添加する。ついで、このゾルを50℃で、2時間保持する。
+16.3ml 0.15M HCl
を添加する。
GMP1、GMP2、GMP3およびGMP4の製造
GMP1、GMP2、GMP3およびGMP4は、下記の条件下で、実施例1に記載の工程で、ゾル1(実施例1)から得た異なる製造ロット由来の色素である:
磁性ガラス粒子を用いたヒト全血由来のPCR試料の前処理
核酸単離
ガラス磁性粒子(GMP2〜4)の3つのロットを各々10mgをエッペンドルフ試験管に移した。厳密な試料の重量を表1に示した。3重の測定を行った。
磁性ガラス粒子への最初の結合後に得られた上清を、その核酸組成について下記のように調べた:該上清を、フィルター試験管(例えば、ハイピュアPCR産物精製キットで供給されるような、ベーリンガーマンハイム社 ID#1744003)に移し、エッペンドルフタブレトップ遠心分離中8000rpmで1分間遠心分離した。フロー−スルー(flow−through)物質を捨て、該フィルター試験管を500μlの洗浄緩衝液で2回洗浄した(前記のように遠心分離)。該フィルター試験管を短時間遠心分離して乾燥し、ついで200μlのあらかじめ70℃に温めておいた1×溶出緩衝液でもう一度遠心分離することによって2回溶出した。
50μlの溶出物およびフィルター試験管を用いて調製した上清に、10μlの試料緩衝液をそれぞれ添加した。45μlのこの調製物を120Vで90分間の電気泳動を用いて0.8%アガロースゲルで分離した。
10秒 92℃
30秒 65℃ 10サイクル
12分 68℃
10秒 92℃
30秒 65℃ 20サイクル
12分プラス 68℃
サイクル当たり20秒
7分 68℃
ついで 7℃
磁性ガラス粒子へのDNA長さ標品の結合
GMP4由来の12mgの磁性ガラス粒子を、エッペンドルフ試験管に移す。
900μlの溶解緩衝液(4.6M GuSCN、45mM Tris、20mM EDTA、pH7.3)およびベーリンガーマンハイム社(Cat.No.528552)のDNA長さ標品IIIがモデルとして添加しされている100μlのDNA試料を、1.5mlエッペンドルフ容器で12mg磁性ガラス粒子と共に2〜10秒間均一な懸濁物が得られるまで混合する。この溶液を室温で20分間インキュベートし、5分ごとに混合する。
磁性フィールド(field)を取り除き、ピペッティングによって800μlの溶液を添加し、2秒間混合し、RTで1分間放置し、該磁性フィールドをアプライし、ついでピペッティングによって上清を取り除くことによって、該磁性ガラス粒子を洗浄緩衝液(5.2M GuSCN、50mM Tris、pH6.5)で2回、70%予冷エタノールで2回、アセトンで1回洗浄する。
繰り返し振盪しながら56℃で10分間インキュベートすることによって、50μlの溶出緩衝液(10mM Tris−HCl、1mM EDTA、pH8.0)で4回DNAを溶出した。ついで、該DNAを含む上清をピペットで新しいエッペンドルフ容器に移す。
試料緩衝液を溶出物量の1/5まで添加し、DNAを90Vの1%アガロースゲル上で分離した。回収を決定するために、試料中に予測されるDNAの量をふくむ同じゲルにDNA長さ標品IIIの希釈シリーズをアプライした。
(1) 一般情報:
(i)出願人:
(A) 名称: ベーリンガー マンハイム ゲーエムベーハー
(B) ストリート: サンドホファー シュトラーセ 116
(C) 市:マンハイム
(E) 国:ドイツ連邦共和国
(F) 郵便番号: 68298
(G) 電話番号: 0621 759 4348
(H) ファクシミリ番号: 0621 759 4457
(ii)発明の名称:磁性色素
(iii)配列の数:2
(iv)コンピュータ可読フォーム:
(A) 媒体タイプ:フレキシブルディスク
(B) コンピュータ:IBM PC 互換機
(C) オペレーティング システム:PC-DOS/MS-DOS
(D) ソフトウェア:パテントイン リリース ♯1.0,バージョン ♯1.30 (EPA)
(2) 配列番号:1の情報:
(i)配列の特徴:
(A) 配列の長さ:34塩基対
(B) 配列の型:核酸
(C) 鎖の数:一本鎖
(D) トポロジー:直鎖状
(ii)配列の種類:他の核酸
(A) 種類: /desc = 「オリゴデオキシリボヌクレオチド」
(iii)ハイポセティカル:NO
(xi)配列:配列番号:1:
ACTGTGCTTC TTGACCCATG GCAGAAGCGC CTTC 34
(2) 配列番号:2の情報:
(i)配列の特徴:
(A) 配列の長さ:34塩基対
(B) 配列の型:核酸
(C) 鎖の数:一本鎖
(D) トポロジー:直鎖状
(ii)配列の種類:他の核酸
(A) 種類: /desc = 「オリゴデオキシリボヌクレオチド」
(iii)ハイポセティカル:NO
(xi)配列:配列番号:2:
CCTTCACTGT CTGCCTAACT CCTTCGTGTG TTCC 34
Claims (9)
- 実質的に無孔または10nm未満の直径の孔を有するガラスの外表面を有してなり、ここで該ガラスは酸化ホウ素を含有する、磁性粒子。
- 粒子が、100μm未満の平均粒径を有することを特徴とする、請求項1記載の磁性粒子。
- 粒子が、10〜60μmの粒径を有することを特徴とする、請求項1または2記載の粒子。
- 表面に存在し得る孔が、1nm未満の直径を有することを特徴とする、請求項1または2記載の粒子。
- 粒子が、強磁性の核を有することを特徴とする、請求項1〜4いずれか記載の粒子。
- 核が、Fe2O3またはFe3O4を含有することを特徴とする、請求項5記載の粒子。
- 粒子が、雲母の核および該雲母に固定された磁性粒子を含有する複合物質を含み、該複合物質はガラス層に埋め込まれていることを特徴とする、請求項1または2記載の粒子。
- a) 磁性核を調製する工程、ならびに、
b) (i) ネットワーク形成成分のアルコキシドを含むアルコール溶液から形成されるゾルを表面上に沈積させる工程、
(ii) 噴霧乾燥過程によりゾル層をゲル層に変換させる工程、および
(iii) 続いてゲルを高密度化させる工程
を含む実質的に無孔のガラス表面で磁性核を包み込む工程
により製造され得る、請求項1〜7いずれか記載の粒子。 - TiO2で被覆された雲母の核および該雲母に固定された磁性粒子からなる複合物質を含み、該複合物質はガラス層に埋め込まれている、請求項1記載の粒子。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19520398A DE19520398B4 (de) | 1995-06-08 | 1995-06-08 | Magnetisches Pigment |
DE19537985A DE19537985A1 (de) | 1995-06-08 | 1995-10-12 | Magnetisches Pigment |
CA002440504A CA2440504C (en) | 1995-06-08 | 1996-06-06 | Magnetic pigment |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP50259397A Division JP3950478B2 (ja) | 1995-06-08 | 1996-06-06 | 磁性色素 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2009238436A Division JP5537894B2 (ja) | 1995-06-08 | 2009-10-15 | 磁性色素 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2006075160A JP2006075160A (ja) | 2006-03-23 |
JP4456046B2 true JP4456046B2 (ja) | 2010-04-28 |
Family
ID=34426455
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP50259397A Expired - Lifetime JP3950478B2 (ja) | 1995-06-08 | 1996-06-06 | 磁性色素 |
JP2005248331A Expired - Lifetime JP4456046B2 (ja) | 1995-06-08 | 2005-08-29 | 磁性色素 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP50259397A Expired - Lifetime JP3950478B2 (ja) | 1995-06-08 | 1996-06-06 | 磁性色素 |
Country Status (14)
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US (3) | US6255477B1 (ja) |
EP (4) | EP0837871B1 (ja) |
JP (2) | JP3950478B2 (ja) |
CN (4) | CN1106401C (ja) |
AT (3) | ATE368109T1 (ja) |
AU (1) | AU707115B2 (ja) |
CA (4) | CA2605671C (ja) |
DE (5) | DE19520398B4 (ja) |
DK (3) | DK1281714T3 (ja) |
ES (3) | ES2197947T3 (ja) |
HK (2) | HK1051048A1 (ja) |
NO (2) | NO325384B1 (ja) |
NZ (1) | NZ311648A (ja) |
WO (1) | WO1996041811A1 (ja) |
Families Citing this family (146)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9425138D0 (en) * | 1994-12-12 | 1995-02-08 | Dynal As | Isolation of nucleic acid |
DE19854973B4 (de) * | 1998-11-30 | 2010-02-04 | Institut Für Neue Materialien Gem. Gmbh | Verfahren zur Reinigung von Nukleinsäuren |
KR100463475B1 (ko) * | 1995-06-08 | 2005-06-22 | 로셰 디아그노스틱스 게엠베하 | 자기성피그먼트 |
DE19520398B4 (de) * | 1995-06-08 | 2009-04-16 | Roche Diagnostics Gmbh | Magnetisches Pigment |
US6545143B1 (en) * | 1998-11-30 | 2003-04-08 | Roche Diagnostics, Gmbh | Magnetic particles for purifying nucleic acids |
JP2965131B2 (ja) | 1995-07-07 | 1999-10-18 | 東洋紡績株式会社 | 核酸結合用磁性担体およびそれを用いる核酸単離方法 |
EP0842208B2 (en) | 1995-07-28 | 2009-08-19 | Sumitomo Chemical Company, Limited | 2,7-aryl-9-substituted fluorenes and 9-substituted fluorene oligomers and polymers |
DE19622885A1 (de) | 1996-06-07 | 1997-12-11 | Boehringer Mannheim Gmbh | Reagenzzubereitung enthaltend magnetische Partikel in Form einer Tablette |
US6027945A (en) | 1997-01-21 | 2000-02-22 | Promega Corporation | Methods of isolating biological target materials using silica magnetic particles |
US20050287583A1 (en) * | 1997-01-21 | 2005-12-29 | Promega Corporation | Methods and kits for isolating biological target materials using silica magnetic particles |
GB9709728D0 (en) | 1997-05-13 | 1997-07-02 | Dynal As | Single step method |
DE19743518A1 (de) * | 1997-10-01 | 1999-04-15 | Roche Diagnostics Gmbh | Automatisierbare universell anwendbare Probenvorbereitungsmethode |
EP1071691B1 (de) | 1998-02-04 | 2005-09-28 | MERCK PATENT GmbH | Verfahren zur isolierung und aufreinigung von nucleinsäuren |
US7078224B1 (en) | 1999-05-14 | 2006-07-18 | Promega Corporation | Cell concentration and lysate clearance using paramagnetic particles |
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