JP6166138B2 - CpGメチル化を用いた同定方法 - Google Patents
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Description
(a)上記サンプル中の特定部位におけるメチル化DNAが存在する際に一貫性のある部位でDNAを切断するメチル活性切断法でサンプルを処理し;
(b)上記特定部位又はその付近においてDNAに相補するプライマーを加え;
(c)上記サンプルをポリメラーゼ連鎖反応に供し、そして当該サンプル中の特定部位においてメチル化DNAが存在する場合、増幅産物を生成し;そして
(d)上記サンプル中の特定部位におけるメチル化DNAの存在を示す増幅産物の存在を検出する
を含む方法に関する。
(a) 上記サンプル中の特定部位におけるメチル化DNAが存在する際に一貫性のある部位で塩基除去をもたらすメチル活性方法でサンプルを処理し;
(b) 切断部位又は塩基除去部位においてDNA配列を変化させ;
(c) 上記特定部位又はその付近においてDNAに相補するプライマー及び/又はプローブを加え、そしてそのうちの少なくとも1つが、変化したDNAを特異的に認識することができる;
(d) 上記サンプルをポリメラーゼ連鎖反応に供し、そしてサンプル中の特定部位においてメチル化DNAが存在する場合、増幅生成物及び/又はプローブシグナルを生成し;そして
(e)増幅生成物又はプローブシグナルが特異的に生成した場合、当該増幅産物の存在を検出し、上記サンプル中の特定部位におけるメチル化DNAの存在を示す
を含む、方法である。
サンプル中の特定部位におけるメチル化DNAが存在する際に一貫性のある部位においてDNAを切断するメチル活性切断方法でサンプルを処理し;
上記特定部位又はその付近においてDNAに相補するプライマーを加え;
上記サンプルをポリメラーゼ連鎖反応に供し、そして上記サンプル中の特定部位においてメチル化DNAが存在する場合、増幅産物を生成し;そして
上記増幅産物の存在を検出し、上記サンプル中の特定部位においてメチル化DNAの存在を示す
方法にさらに関する。
「アンプリコン」は、ポリメラーゼ連鎖反応などの増幅反応の結果として生成した二本鎖DNA分子を指す。
本発明は、メチル活性切断の幾つかの方法を包含する。メチル化CpGが存在する際にDNAを切断するが、メチル化DNAが存在しない際にDNAを切断できない任意の方法が、本発明において使用することができる。本発明の方法は、メチル活性制限酵素、例えばMcrBC(Stewart, F. J. and Raleigh E. A. (1998) Biol. Chem. 379: 611-616)及びリアーゼと組み合わせた5-メチルシチジングリコシラーゼを含むが、それらに限定されるものではない。
様々な増幅方法が、本発明により想定されており、例えばポリメラーゼ連鎖反応(PCR)、リガーゼ連鎖反応、及びローリングサイクル複製が挙げられるがこれらに限定されるものではない。
増幅生成物の特定種を検出する幾つかの方法が、本発明により検討される。検出方法としては、標識の取り込みと検出、プローブキャプチャー法、Taqmanアッセイ、電気泳動法、及びハイブリダイゼーション方法が挙げられるが、それらに限定されるものではない。標識は、放射性標識ヌクレオチド、フルオロフォア、量子ドット、ビオチンコンジュゲートヌクレオチド、及び発色酵素が挙げられるが、それらに限定されるものではない。
以下の記載は予言的であり、実際の実験を表すものではない。
実施例1
液体サンプルを患者から採取することができる。ゲノムDNAが、既知の方法を用いて患者サンプルから抽出することもできる。
さらなる実施例では、固形腫瘍生検を患者から取得する。確立された技術を用いて、固形腫瘍生検からゲノムDNAを抽出して、サンプルを生成する。
さらなる実施例では、固形腫瘍生検は、患者から得ることができる。確立された技術を用いて、固形腫瘍生検からゲノムDNAを抽出し、サンプルを生成する。
Claims (3)
- 患者由来のサンプル中のゲノムDNAの特定部位においてメチル化DNAを検出する方法であって、以下の:
上記ゲノムDNAを、メチル活性制限酵素で処理して、切断生成物を作成する工程、
上記切断生成物に平滑末端を作成する工程、
上記切断生成物の末端をライゲーションして、当該切断生成物の閉環を作成する工程、
上記切断生成物に対して、上記特定部位又はその付近のDNAに相補するプライマーを加える工程であって、上記増幅生成物が上記切断生成物のライゲーションされた閉環からのみ生じうるように、プライマーを配向させる工程、
上記サンプルをポリメラーゼ連鎖反応に供して、上記特定部位においてメチル化DNAが存在する場合に増幅生成物を取得する工程、及び
上記特定部位においてメチル化DNAの存在を示す増幅生成物の存在を検出する工程 を含む、前記方法。 - 前記特定部位が、遺伝子のプロモーター領域である、請求項1に記載の方法。
- 前記メチル活性制限酵素が、大腸菌(E.coli)McrBCである、請求項1に記載の方法。
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CA (1) | CA2694820C (ja) |
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US8709762B2 (en) | 2010-03-02 | 2014-04-29 | Bio-Rad Laboratories, Inc. | System for hot-start amplification via a multiple emulsion |
US8951939B2 (en) | 2011-07-12 | 2015-02-10 | Bio-Rad Laboratories, Inc. | Digital assays with multiplexed detection of two or more targets in the same optical channel |
US9417190B2 (en) | 2008-09-23 | 2016-08-16 | Bio-Rad Laboratories, Inc. | Calibrations and controls for droplet-based assays |
WO2011120020A1 (en) | 2010-03-25 | 2011-09-29 | Quantalife, Inc. | Droplet transport system for detection |
WO2011120006A1 (en) | 2010-03-25 | 2011-09-29 | Auantalife, Inc. A Delaware Corporation | Detection system for droplet-based assays |
WO2011120024A1 (en) | 2010-03-25 | 2011-09-29 | Quantalife, Inc. | Droplet generation for droplet-based assays |
US9764322B2 (en) | 2008-09-23 | 2017-09-19 | Bio-Rad Laboratories, Inc. | System for generating droplets with pressure monitoring |
US8633015B2 (en) | 2008-09-23 | 2014-01-21 | Bio-Rad Laboratories, Inc. | Flow-based thermocycling system with thermoelectric cooler |
US9132394B2 (en) | 2008-09-23 | 2015-09-15 | Bio-Rad Laboratories, Inc. | System for detection of spaced droplets |
US10512910B2 (en) | 2008-09-23 | 2019-12-24 | Bio-Rad Laboratories, Inc. | Droplet-based analysis method |
US9492797B2 (en) | 2008-09-23 | 2016-11-15 | Bio-Rad Laboratories, Inc. | System for detection of spaced droplets |
US9598725B2 (en) | 2010-03-02 | 2017-03-21 | Bio-Rad Laboratories, Inc. | Emulsion chemistry for encapsulated droplets |
US9156010B2 (en) | 2008-09-23 | 2015-10-13 | Bio-Rad Laboratories, Inc. | Droplet-based assay system |
US11130128B2 (en) | 2008-09-23 | 2021-09-28 | Bio-Rad Laboratories, Inc. | Detection method for a target nucleic acid |
EP2473618B1 (en) | 2009-09-02 | 2015-03-04 | Bio-Rad Laboratories, Inc. | System for mixing fluids by coalescence of multiple emulsions |
EP2635840B1 (en) | 2010-11-01 | 2017-01-04 | Bio-Rad Laboratories, Inc. | System for forming emulsions |
CA2830443C (en) | 2011-03-18 | 2021-11-16 | Bio-Rad Laboratories, Inc. | Multiplexed digital assays with combinatorial use of signals |
EP3789498A1 (en) | 2011-04-25 | 2021-03-10 | Bio-rad Laboratories, Inc. | Methods for nucleic acid analysis |
EP2737089B1 (en) | 2011-07-29 | 2017-09-06 | Bio-rad Laboratories, Inc. | Library characterization by digital assay |
WO2013155531A2 (en) | 2012-04-13 | 2013-10-17 | Bio-Rad Laboratories, Inc. | Sample holder with a well having a wicking promoter |
CN103233072B (zh) * | 2013-05-06 | 2014-07-02 | 中国海洋大学 | 一种高通量全基因组dna甲基化检测技术 |
CN104357573B (zh) * | 2014-11-19 | 2016-09-28 | 苏州圣苏新药开发有限公司 | 精确验证未知基因甲基化程度的方法 |
US20200399678A1 (en) * | 2017-10-30 | 2020-12-24 | Naomi Yamakawa | Method for analyzing dna methylation using next generation sequencer and method for concentrating specific dna fragments |
GB201810571D0 (en) * | 2018-06-27 | 2018-08-15 | Cs Genetics Ltd | Reagents and methods for the analysis of circulating microparticles |
WO2024083982A1 (en) * | 2022-10-21 | 2024-04-25 | F. Hoffmann-La Roche Ag | Detection of modified nucleobases in nucleic acid samples |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1487999B1 (en) * | 2002-03-15 | 2006-12-27 | Epigenomics AG | Discovery and diagnostic methods using 5-methylcytosine dna glycosylase |
EP1723256B1 (en) * | 2004-02-18 | 2010-04-14 | Centre for Addiction and Mental Health | CpG-AMPLICON AND ARRAY PROTOCOL |
CA2902980A1 (en) * | 2004-03-08 | 2005-09-29 | Rubicon Genomics, Inc. | Methods and compositions for generating and amplifying dna libraries for sensitive detection and analysis of dna methylation |
WO2008143936A1 (en) | 2007-05-18 | 2008-11-27 | Schering Corporation | Methods of enhancing adenoviral delivery |
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CN101765666B (zh) | 2015-04-29 |
WO2009015863A2 (en) | 2009-02-05 |
HK1141837A1 (en) | 2010-11-19 |
CA2694820C (en) | 2015-06-16 |
CA2694820A1 (en) | 2009-02-05 |
EP2176428B1 (en) | 2017-04-12 |
WO2009015863A4 (en) | 2009-07-16 |
JP5844522B2 (ja) | 2016-01-20 |
US20110136120A1 (en) | 2011-06-09 |
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EP2176428A2 (en) | 2010-04-21 |
WO2009015863A3 (en) | 2009-04-23 |
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