JP5336087B2 - 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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- JP5336087B2 JP5336087B2 JP2007553214A JP2007553214A JP5336087B2 JP 5336087 B2 JP5336087 B2 JP 5336087B2 JP 2007553214 A JP2007553214 A JP 2007553214A JP 2007553214 A JP2007553214 A JP 2007553214A JP 5336087 B2 JP5336087 B2 JP 5336087B2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0636—Integrated biosensor, microarrays
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- B01L2300/08—Geometry, shape and general structure
- B01L2300/0896—Nanoscaled
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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 (8)
- (a)入口ポートと、
(b)出口ポートと、
(c)上記入口ポートおよび上記出口ポートの中間にあり、上記入口ポートおよび上記出口ポートと液体連通する単一の結合チャネルとを有し、
上記チャネルの表面の少なくとも一部が、核酸を単離し精製するために有効な未改質の平坦ガラス表面であり、上記結合チャネルは、矩形の断面を有し、頂壁、底壁、および2つの側壁を有する装置。 - 上記核酸は二本鎖DNAまたは一本鎖DNAまたはRNAである請求項1記載の装置。
- 上記核酸を計量する手段であって蛍光読み取り手段を具備するものをさらに有する請求項1または2記載の装置。
- カオトロピック塩溶液から核酸を補足する方法であって、核酸を含有するカオトロピック塩溶液を請求項1記載の装置に通し、上記核酸を含有する上記溶液が上記未改質の平滑なガラス表面に接触させて固定化された核酸を取得するステップを有する上記方法。
- 固定化された上記核酸は二本鎖DNAまたは一本鎖DNAまたはRNAである請求項4記載の方法。
- 固定された上記核酸を洗浄するステップをさらに有する請求項4または5記載の方法。
- 固定された上記核酸を上記ガラス表面から開放して開放された核酸を取得するステップをさらに有する請求項4〜6のいずれかに記載の方法。
- 開放された上記核酸を計量するステップをさらに有する請求項7記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/043,561 | 2005-01-26 | ||
US11/043,561 US20060166223A1 (en) | 2005-01-26 | 2005-01-26 | DNA purification and analysis on nanoengineered surfaces |
PCT/US2006/002708 WO2006081324A2 (en) | 2005-01-26 | 2006-01-25 | Dnd purification and analysis on nanoengineered surfaces |
Publications (3)
Publication Number | Publication Date |
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JP2008532485A JP2008532485A (ja) | 2008-08-21 |
JP2008532485A5 JP2008532485A5 (ja) | 2009-03-26 |
JP5336087B2 true JP5336087B2 (ja) | 2013-11-06 |
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Application Number | Title | Priority Date | Filing Date |
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JP2007553214A Expired - Fee Related JP5336087B2 (ja) | 2005-01-26 | 2006-01-25 | ナノ加工表面におけるdna浄化および分析 |
Country Status (9)
Country | Link |
---|---|
US (2) | US20060166223A1 (ja) |
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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-
2005
- 2005-01-26 US US11/043,561 patent/US20060166223A1/en not_active Abandoned
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2006
- 2006-01-25 ES ES06719536T patent/ES2397352T3/es active Active
- 2006-01-25 AU AU2006208078A patent/AU2006208078A1/en not_active Abandoned
- 2006-01-25 CA CA002595268A patent/CA2595268A1/en not_active Abandoned
- 2006-01-25 WO PCT/US2006/002708 patent/WO2006081324A2/en active Application Filing
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- 2006-01-25 EP EP06719536A patent/EP1851337B1/en not_active Not-in-force
- 2006-01-25 JP JP2007553214A patent/JP5336087B2/ja not_active Expired - Fee Related
- 2006-01-25 CN CNA2006800097994A patent/CN101248187A/zh active Pending
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CA2595268A1 (en) | 2006-08-03 |
ES2397352T3 (es) | 2013-03-06 |
EP1851337A2 (en) | 2007-11-07 |
CN101248187A (zh) | 2008-08-20 |
WO2006081324A3 (en) | 2007-11-22 |
US20060166223A1 (en) | 2006-07-27 |
US20090029867A1 (en) | 2009-01-29 |
EP1851337A4 (en) | 2009-03-11 |
WO2006081324A2 (en) | 2006-08-03 |
KR20080047311A (ko) | 2008-05-28 |
AU2006208078A1 (en) | 2006-08-03 |
EP1851337B1 (en) | 2012-10-10 |
JP2008532485A (ja) | 2008-08-21 |
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