WO2014025001A1 - 核酸配列の切断標識方法 - Google Patents
核酸配列の切断標識方法 Download PDFInfo
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- WO2014025001A1 WO2014025001A1 PCT/JP2013/071576 JP2013071576W WO2014025001A1 WO 2014025001 A1 WO2014025001 A1 WO 2014025001A1 JP 2013071576 W JP2013071576 W JP 2013071576W WO 2014025001 A1 WO2014025001 A1 WO 2014025001A1
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- Prior art keywords
- nucleic acid
- target nucleic
- acid sequence
- labeled
- fluorescence
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6841—In situ hybridisation
Definitions
- two or more types of probe DNAs labeled with fluorescent dyes that emit fluorescent colors with different color tones are used, and they are simultaneously fluorescent. There is something that maps chromosomes.
- the present invention when using the FISH method or the CISH method or the like, when observing the color of the fluorescence or coloring regardless of the relative position and angle between the fluorescence microscope and the target DNA.
- Another object of the present invention is to provide a method for cleaving a target nucleic acid, which can prevent an uncut target DNA from being mistaken for being cleaved.
- the second aspect in addition to the effects obtained in the first aspect, it can be determined more clearly whether the nucleic acid sequence is cleaved at the cleavage region or at other sites.
- the “important part” refers to a part of a nucleic acid sequence that is determined to be important by a person who carries out the present invention.
- the present invention when the present invention is applied to a cancer diagnosis method, It refers to a part of an important nucleic acid sequence (for example, kinase domain).
- an important nucleic acid sequence for example, kinase domain.
- at least one important part of the nucleic acid sequence is sufficient, and two or more important parts may be present in the nucleic acid sequence.
- the present invention may be carried out with a person who implements the present invention more important as an important part of the present invention.
- the hybridization solution used in the first step is composed of two or more labeled probes labeled with different identification factors, and includes a probe set that can hybridize to almost the entire sequence of the target nucleic acid. There is no particular limitation.
- the yellow fluorescence of the mixing portion ⁇ can be observed together with the orange fluorescence and the green fluorescence. Therefore, by confirming them, it can be more reliably determined whether the target nucleic acid 1 is cleaved at the breakpoint 7 included in the cleavage region 2 or is cleaved elsewhere. (Embodiment 3)
- the probe set 45 is configured in this manner, the same effect as in the case of the probe set 5 of Embodiment 1 can be obtained, and the labeled probe 45b has the same length as the target nucleic acid 1; Measure the intensity of fluorescence of the green fluorescent dye labeled on the green fluorescent dye of the target nucleic acid 1 when not cleaved, and the intensity of the green fluorescent dye of the target nucleic acid 1 when cleaved By comparing the intensity of fluorescence, the cleavage position of the target nucleic acid 1 can be estimated more accurately than in the case of the probe set 5 of Embodiment 1. (Embodiment 4)
- RP11-984I21, RP11-62B19 and RP11-701P18 labeled with FITC as labeled probes, and then the conventional FISH method A fluorescent image was created by performing the same operation as described above.
- RP11-984I21, RP11-62B19 and RP11-701P18 are identification numbers in the human male RP-11RP BAC library.
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- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
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- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
(実施形態1)
(実施形態2)
(実施形態3)
(実施形態4)
(他の実施形態)
1A,1B,1A’,1B’ 標的核酸片
2, 102 切断領域
3, 103 重要部
7,8,107,108 切断点
5,15,25,35,45,55,65,75,85,105 プローブセット
5A,5B,5A’,5B’,25A,25B プローブセット片
5a,5b,5a’,5b’,15a,15b,25a,25b,35a,45a,45b,55b,65a,75b,85a,85b,85c,105a,105b 標識プローブ
α 混合部
Claims (9)
- 少なくとも1つの切断点を含む切断領域を有する標的核酸配列が切断されているか否かを判別する核酸配列の切断標識方法であって、
異なる識別因子で標識された2つ以上の標識プローブからなり、当該標的核酸のほぼ全配列とハイブリダイゼーションできるプローブセットを含むハイブリダイゼーション液と、当該標的核酸と、を接触させる工程と、
当該識別因子を機能させてシグナルを出させる工程と、
を有することを特徴とする核酸配列の切断標識方法。 - 前記標識プローブの少なくとも1つが前記核酸配列の切断領域にハイブリダイゼーションすることを特徴とする請求項1に記載の核酸配列の切断標識方法。
- 前記核酸配列は少なくとも1つの重要部をさらに有し、
前記核酸配列の切断領域にハイブリダイゼーションする標識プローブと、
前記切断領域に対して前記重要部を挟むように位置する前記核酸配列の部分にハイブリダイゼーションする標識プローブとが、異なる標識プローブであること
を特徴とする請求項2に記載の核酸配列の切断標識方法。 - 前記プローブセットが異なる識別因子が混ざった状態で標識された混合部を有することを特徴とする請求項1乃至3のいずれかに記載の核酸配列の切断標識方法。
- 前記混合部が前記重要部の一部または全部とハイブリダイゼーションすることを特徴とする請求項4に記載の核酸配列の切断標識方法。
- 同じ識別因子で標識された単独または複数の標識プローブが当該標的核酸のほぼ全配列とハイブリダイゼーションすることを特徴とする請求項1乃至5のいずれかに記載の核酸配列の切断標識方法。
- 前記異なる識別因子が異なる色の色素であり、
前記識別因子を機能させてシグナルを出させる工程が当該色素に光を照射して発色させる工程であり、
当該シグナルを検出する工程が当該発色を検出する工程であること
を特徴とする請求項1から6のいずれかに記載の核酸配列の切断標識方法。 - 前記異なる識別因子が異なる蛍光色を発する蛍光色素であり、
前記識別因子を機能させてシグナルを出させる工程が当該蛍光色素を蛍光させる工程であり、
当該シグナルを検出する工程が当該蛍光を検出する工程であること
を特徴とする請求項1から6のいずれかに記載の核酸配列の切断標識方法。 - 前記色素が修正マンセル修正表色系において2つの異なる色相の発色するものまたは蛍光色素が修正マンセル修正表色系において2つの異なる色相の蛍光色を発するものからなり、
前記切断点が、当該異なる色相のうち、当該色相と修正マンセル色相環の中心点とを結ぶ直線と、当該2つの異なる色相を混色させた際の当該混色の色相と修正マンセル色相環の中心点とを結ぶ直線とがなす角の大きい方の色相の色素または蛍光色素で標識されている部分に対応する核酸配列にあること
を特徴とする請求項7または8に記載の核酸配列の切断標識方法。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014529569A JP6067020B2 (ja) | 2012-08-09 | 2013-08-08 | 核酸配列の切断標識方法 |
EP13828279.3A EP2883964A4 (en) | 2012-08-09 | 2013-08-08 | METHOD FOR NUCLEIC ACID SEQUENCE CLEAVAGE MARKING |
US14/420,635 US20150322498A1 (en) | 2012-08-09 | 2013-08-08 | Method for labelling of cleavage of nucleic acid sequence |
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JP2012176558 | 2012-08-09 | ||
JP2012-176558 | 2012-08-09 |
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WO2014025001A1 true WO2014025001A1 (ja) | 2014-02-13 |
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PCT/JP2013/071576 WO2014025001A1 (ja) | 2012-08-09 | 2013-08-08 | 核酸配列の切断標識方法 |
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US (1) | US20150322498A1 (ja) |
EP (1) | EP2883964A4 (ja) |
JP (1) | JP6067020B2 (ja) |
WO (1) | WO2014025001A1 (ja) |
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EP4092136B8 (en) * | 2021-05-20 | 2024-03-13 | Sophia Genetics S.A. | Capture probes and uses thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2002542793A (ja) | 1999-04-22 | 2002-12-17 | ザ アルバート アインシュタイン カレッジ オブ メディシン オブ イエシバ ユニバーシティ | 多蛍光fishによる遺伝子発現パターンのアッセイ法 |
JP2004157053A (ja) | 2002-11-07 | 2004-06-03 | Japan Science & Technology Agency | 二重染色法によるガン診断方法 |
JP2007535966A (ja) * | 2004-05-04 | 2007-12-13 | ダコ デンマーク アクティーゼルスカブ | 染色体異常を検出するための方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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EP0878552A1 (en) * | 1997-05-13 | 1998-11-18 | Erasmus Universiteit Rotterdam | Molecular detection of chromosome aberrations |
US6414133B1 (en) * | 1998-10-13 | 2002-07-02 | Ventana Medical Systems, Inc. | Multiple fusion probes |
CA2447320A1 (en) * | 2001-05-14 | 2002-11-21 | Cancer Genetics, Inc. | Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish) |
JP5861244B2 (ja) * | 2010-06-22 | 2016-02-16 | 公益財団法人がん研究会 | 新規ros1融合体の検出法 |
-
2013
- 2013-08-08 EP EP13828279.3A patent/EP2883964A4/en not_active Ceased
- 2013-08-08 JP JP2014529569A patent/JP6067020B2/ja active Active
- 2013-08-08 US US14/420,635 patent/US20150322498A1/en not_active Abandoned
- 2013-08-08 WO PCT/JP2013/071576 patent/WO2014025001A1/ja active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2002542793A (ja) | 1999-04-22 | 2002-12-17 | ザ アルバート アインシュタイン カレッジ オブ メディシン オブ イエシバ ユニバーシティ | 多蛍光fishによる遺伝子発現パターンのアッセイ法 |
JP2004157053A (ja) | 2002-11-07 | 2004-06-03 | Japan Science & Technology Agency | 二重染色法によるガン診断方法 |
JP2007535966A (ja) * | 2004-05-04 | 2007-12-13 | ダコ デンマーク アクティーゼルスカブ | 染色体異常を検出するための方法 |
Non-Patent Citations (1)
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Also Published As
Publication number | Publication date |
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EP2883964A4 (en) | 2016-04-27 |
JPWO2014025001A1 (ja) | 2016-07-25 |
US20150322498A1 (en) | 2015-11-12 |
EP2883964A1 (en) | 2015-06-17 |
JP6067020B2 (ja) | 2017-01-25 |
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