JP2008532485A - ナノ加工表面におけるdna浄化および分析 - Google Patents
ナノ加工表面におけるdna浄化および分析 Download PDFInfo
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Abstract
【選択図】 図3
Description
この発明はSBIR許諾番号GM072178−01の下で政府の支援の下でなされた。政府はこの発明に関し所定の権利を有するかもしれない。
[関連出願の相互参照]
この出願は2005年1月26日に提出された米国特許出願第11/043,561により生じる利益を要求し、その出願の内容は参照してここに組み入れる。
例えば、H33258のブロモアセチル類似物はスルフヒドリル改質オリゴヌクレオチドと共役であった。高感度のDNA結合分析がシアニン型の染料の蛍光特性に基づいて開発されてきた。TOもRNAの存在下で3000倍までの増大を示す。この特性は興味深いものであり、RNA単離装置に適用できる。
1.高濃度のGuSCNを用いて細胞を溶解し、ヒストンを除去し、DNAをガラスに結合する。
2.ガラス結合DNAを高濃度GuSCNで洗浄する。
3.生理食塩で結合DNAをガラスから開放する。
4.蛍光性基体を用いてチャンバー内の開放DNAの濃度を読み取る。
5.DNAを蛍光性基体から開放してPCRベースの分析に使用するために調整する。
結合ヘキシルアミンがリンクされた蛍光性Hoechst染料の合成が実行された。ここで図11を参照すると、合成が図示される。とくに断らない限り、試薬はSigma−Adrichから取得した。無水性溶媒が確実に封止されたビン中に取得された。7cm長のTLCストリップを5×7cmのシリカ被覆アルミニウムシート(Merck)から切り出し、手持ち長波長UVランプを用いて照射して蛍光により全般的に視覚化した。溶媒前面(Rf)に対する蛍光スポットの移動をTLC分析のために測定した。蛍光性DNA結合分析用に用いられるHoechst33258標準(ビスベンズイミド、BB−OH)は、Sigma社から販売されているDNA Quantitation Kit中に取得した。このキットはビスベンズイミド(水中で10mg/mL)、10倍蛍光分析緩衝剤(10×FAB=100mMのトリス塩酸、10mMのEDTA、2MのNaCl、pH7.4)、およびDNA標準(刈り込まれたcarf胸腺DNA、1×FAB中に1mg/mL)を含む。このキットは、図12に示されるDNAの蛍光性測定の標準測定として用いられた。
1.インジケータ:
dsDNAの存在下でのみ蛍光性である。
リンカーはほとんど蛍光性を持たない背景とともにスペーサ分子に結合する。
2.基体:
関与対象のスペクトル領域で透明である。
スペーサ/インジケータ分子で簡単に被覆される。
3.スペーサ:
蛍光性インジケータおよび基体に対して簡易に共役化される。
蛍光性MBを立体障害を伴うことなくdsDNAにアクセス可能にする。
1.高濃度のGuSCNを用いて、細胞を溶解し、ヒストンを除去し、DNAをガラスに結合する。
2.ガラス結合DNAを高濃度GuSCNで洗浄する。
3.生理食塩で結合DNAをガラスから開放する。
4.蛍光性基体を用いてチャンバー内の開放DNAの濃度を読み取る。
5.DNAを蛍光性基体から開放してPCRベースの分析に使用するために調整する。
52、58 小さな粒子
54 血液全体
56 第2の流れ
60 純粋な溶液
62 共通チャネル
Claims (20)
- 流体入口ポートと、
上記流体入口ポートに接続されたDNA結合チャネルと、
流体出力ポートとを有し、
上記DNA結合チャネル内の少なくとも1つの表面の少なくとも一部が、DNAが特異的に上記表面に結合するような結合試薬により改質されたことを特徴とするマイクロ流体システム。 - 上記結合チャネルは、基本的に矩形の断面を有し、頂壁、底壁、および2つの側壁を有する請求項1記載のマイクロ流体システム。
- 上記結合チャネルは頂壁および底壁の間に200マイクロメータの距離を有し、結合試薬により改質された上記DNA結合チャネル内の上記表面は上記頂壁または上記底壁、または上記頂壁および上記底壁に位置する請求項2記載のマイクロ流体システム。
- 上記流体入口ポートは、ピペット先端用の圧縮シールを実現する請求項1記載の装置。
- 少なくとも1つの壁は他の壁と異なる材料で製造される請求項2記載の装置。
- 上記少なくとも1つの壁はガラスで製造される請求項2記載の装置。
- 上記DNA結合チャネルに結合された廃棄物貯蔵チャネルをも有する請求項2記載の装置。
- 上記DNA結合チャネルに結合された生成物ウェルをも有する請求項2記載の装置。
- 上記DNA結合チャネルに結合された生成物ウェルをも有する請求項2記載の装置。
- 上記装置は、上記装置内に含まれる染料分子を励起する光、および、上記染料分子から発光される光を透過させることができる材料から製造される請求項1記載の装置。
- 96ウェルプレートリーダー中での配置および読み出しに適合化されたサイズおよび幾何形状を有する請求項1記載の装置。
- ポリマー性DNAおよりRNAの濃度を決定するための、固定化された核酸(NA)指示蛍光分子を有する組成物。
- 蛍光測定用の所望の光学特性を具備するNA指示分子を結合可能な基体をさらに有する請求項12記載の組成物。
- 上記NA指示分子は二重螺旋DNAまたは単一螺旋DNAに特異的である請求項12記載の組成物。
- 上記NA指示分子はベンズイミダゾール染料(Hoechst33258)およびDNAターゲットシアニン染料(ビス−シアニン染料)を含むグループから採用される請求項14記載の組成物。
- 上記NA指示分子はRNAに対して特異的であり、チアゾールオレンジを含むグループから採用される請求項14記載の組成物。
- 流体入口ポートと、
上記流体入口ポートに接続されたDNA結合チャネルと、
流体出力ポートとを有し、
上記DNA結合チャネル内の少なくとも1つの表面の少なくとも一部が、DNAが特異的に上記表面に結合するような結合試薬により改質され、上記結合試薬はポリマー性DNAまたはRNAの濃度を決定するための、固定化された核酸(NA)指示蛍光分子を有することを特徴とするマイクロ流体システム。 - 核酸を単離し精製するための未改質の表面をも有する請求項17記載の装置。
- 上記基体は、ガラス、プラスチック、ニトロセルロース、金属、または他のポリマー材料からなるグループから採用される請求項13記載の組成物。
- 上記基体は、上記NA指示蛍光分子中のアミド基と結合可能な活性化エステル基またはシアヌル酸残余のような求電子基により活性化されたガラススライドを有する請求項13記載の組成物。
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US11/043,561 US20060166223A1 (en) | 2005-01-26 | 2005-01-26 | DNA purification and analysis on nanoengineered surfaces |
US11/043,561 | 2005-01-26 | ||
PCT/US2006/002708 WO2006081324A2 (en) | 2005-01-26 | 2006-01-25 | Dnd purification and analysis on nanoengineered surfaces |
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JP2008532485A true JP2008532485A (ja) | 2008-08-21 |
JP2008532485A5 JP2008532485A5 (ja) | 2009-03-26 |
JP5336087B2 JP5336087B2 (ja) | 2013-11-06 |
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EP (1) | EP1851337B1 (ja) |
JP (1) | JP5336087B2 (ja) |
KR (1) | KR20080047311A (ja) |
CN (1) | CN101248187A (ja) |
AU (1) | AU2006208078A1 (ja) |
CA (1) | CA2595268A1 (ja) |
ES (1) | ES2397352T3 (ja) |
WO (1) | WO2006081324A2 (ja) |
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JP2012508015A (ja) * | 2008-11-04 | 2012-04-05 | ブラッド・セル・ストレイジ,インコーポレイテッド | 湾曲したガラス表面上での核酸抽出 |
JP2013117424A (ja) * | 2011-12-02 | 2013-06-13 | Enplas Corp | 流体取扱装置 |
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US20060166223A1 (en) * | 2005-01-26 | 2006-07-27 | Reed Michael W | DNA purification and analysis on nanoengineered surfaces |
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DK2097541T3 (da) * | 2006-10-10 | 2013-03-18 | Miacom Diagnostics Gmbh | Nukleinsyre-beacons til fluorescent in-situ hybridisering og chip-teknologi |
WO2008109878A2 (en) * | 2007-03-07 | 2008-09-12 | California Institute Of Technology | Testing device |
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WO2009117167A1 (en) * | 2008-01-02 | 2009-09-24 | Blood Cell Storage, Inc. | Devices and processes for nucleic acid extraction |
DE102008047790A1 (de) * | 2008-09-17 | 2010-04-15 | Qiagen Gmbh | Verfahren zur Normierung des Gehalts von Biomolekülen in einer Probe |
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AU2006208078A1 (en) | 2006-08-03 |
EP1851337A2 (en) | 2007-11-07 |
WO2006081324A2 (en) | 2006-08-03 |
US20090029867A1 (en) | 2009-01-29 |
CA2595268A1 (en) | 2006-08-03 |
KR20080047311A (ko) | 2008-05-28 |
ES2397352T3 (es) | 2013-03-06 |
US20060166223A1 (en) | 2006-07-27 |
WO2006081324A3 (en) | 2007-11-22 |
EP1851337B1 (en) | 2012-10-10 |
EP1851337A4 (en) | 2009-03-11 |
CN101248187A (zh) | 2008-08-20 |
JP5336087B2 (ja) | 2013-11-06 |
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