JP2019536435A - スモールrna又はスモールrnaに関連するタンパク質を検出する方法 - Google Patents
スモールrna又はスモールrnaに関連するタンパク質を検出する方法 Download PDFInfo
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Abstract
Description
0個の塩基で構成されたとき、少なくとも3個以上、好ましくは3〜10個、より好ましくは3〜9個、さらにより好ましくは3〜8個、さらにより好ましくは3〜7個、さらにより好ましくは3〜6個、さらにより好ましくは3〜5個、さらにより好ましくは3〜4個の塩基配列で構成される。また、前記Yは、Zが1個の塩基で構成されたとき、少なくとも2個以上、好ましくは2〜10個、より好ましくは2〜9個、さらにより好ましくは2〜8個、さらにより好ましくは2〜7個、さらにより好ましくは2〜6個、さらにより好ましくは2〜5個、さらにより好ましくは2〜4個、さらにより好ましくは2〜3個の塩基配列で構成された。Yを構成する塩基配列が前記範囲を外れた場合には、核酸又はタンパク質のリアルタイム検出時に、非特異的反応が発生して感度が低下する可能性がある。
ter 556、Oyster 645、Bodipy 630/650、Bodipy
650/665、Calfluor ORNAge 546、Calfluor red 610、 Quasar 670およびビオチンからなる群より選ばれるいずれか1つであることができるが、必ずしもこれらに限定されるものではない。また、前記消光物質は、例えば、DDQ−1、Dabcyl、Eclipase、6−TAMRA、BHQ−1、BHQ−2、BHQ−3、lowa Black RQ−Sp、QSY−7、QSY−2およびMGBNFQからなる群より選ばれるいずれか1つであることができるが、必ずしもこれらに限定されるものではない。
特異的に切断できるものであればいかなるものであってもよい。例えば、Y部位がDNAである場合は、DNAヌクレアーゼ(DNA nuclease、DNase)、具体的には、DNase I、DNase II、S1核酸加水分解酵素、核酸加水分解酵素P1、APエンドヌクレアーゼ、又はUvrABSC核酸加水分解酵素などを使用することが好ましく、Y部位がRNAである場合は、RNA加水分解酵素(ribonuclease、RNase)、具体的には、RNase II、RNase III、RNase IV、RNase H、又はRNase T2等を使用することが好ましい。
I、DNase II、S1核酸加水分解酵素、核酸加水分解酵素P1、APエンドヌクレアーゼ、又はUvrABSC核酸加水分解酵素などを使用することが好ましく、X部位がRNAである場合は、RNA加水分解酵素(ribonuclease、RNase)、具体的には、RNase II、RNase III、RNase IV、RNase H、又はRNase T2などを使用することが好ましい。
する蛍光物質、又は蛍光物質および消光物質からなる蛍光ペアを用いることができる。
650/665、Calfluor ORNAge 546、Calfluor red 610、 Quasar 670およいビオチンからなる群より選ばれるいずれか1つであり得るが、 必ずしもこれらに限定されるものではない。また、前記消光物質は、例えば、DDQ−1、Dabcyl、Eclipase、6−TAMRA、BHQ−1、BHQ−2、BHQ−3、lowa Black RQ−Sp、QSY−7、QSY−2およびMGBNFQからなる群より選ばれるいずれか1つであってもよく、必ずしもこれらに限定されるものではない。
acement amplification)および核酸配列ベースの増幅(nucleic acid sequence based amplification)からなる群より選ばれるいずれか1つの方法により行われるが、必ずしもこれらに限定されるものではない。
増幅反応により合成されたものである。
a)、miRNA−155(5’−uua aug cua auc gug aua
ggg gu)の遺伝子発現を測定するために、本発明に係る一本鎖核酸(promo
r)とプライマー(primer)、RTプライマーを、下記表1に示すように、IDT(Integrated DNA Technologies、USA)に依頼して調製した(表1参照)。ここで、本発明に係る一本鎖核酸(promor)の場合、5’末端には、FAM(fluorescein succinimidyl ester)を、3’末端には3IABkFGを付着しており、リボヌクレオチド(RNA)は、デオキシリボヌクレオチド(DNA)との区別のために、配列の前に添え字「r」で示した。
に希釈した。
Claims (10)
- a)生物学的試料から検出しようとするスモールRNA(small RNA)を含むRNAを得る工程と、b)前記a)工程で得られたRNAから検出しようとするスモールRNAの一部の塩基配列と相補的結合が可能な塩基配列が含まれたX−Y−Z構造の一本鎖核酸を用いてcDNAを合成する工程と、c)前記b)工程のcDNAを増幅する工程と、d)前記b)工程の前記一本鎖核酸のY部位を切断可能な酵素により切断された一本鎖核酸断片の量を測定する工程と、を含み、
又は、a−1)生物学的試料から検出しようとするスモールRNA(small RNA)を含むRNAを得る工程と、b−1)前記a−1)工程で得られたRNAからcDNAを合成する工程と、c−1)前記b−1)工程のcDNAに前記cDNAの一部の塩基配列と相補的結合が可能な塩基配列を含み、(i)フォワードプライマー及びプローブ、(ii)リバースプライマー及びプローブ、又は(iii)フォワードプライマーとリバースプライマーとプローブとして使用可能な一本鎖核酸を混合して、前記cDNAを増幅する工程と、d−1)前記c−1)工程の前記一本鎖核酸のY部位を切断可能な酵素により切断された一本鎖核酸断片の量を測定する工程と、を含むスモールRNA(small RNA)を検出する方法であって、
前記一本鎖核酸はX−Y−Zの構造を有し、前記一本鎖核酸の両末端又は内部に1つ又はそれ以上の検出可能なマーカーが付されており、リアルタイム検出を目的とする標的核酸又は標的タンパク質の特定の部位に結合して複合体を形成し、特定の酵素によりY部位の切断時に、X部位は、標的核酸又は標的タンパク質と複合体を維持しながらプライマーとして作用し、Y及びZは、前記標的核酸又は標的タンパク質の特定の部位から分離されることを特徴とし、
前記X、Y、及びZは、塩基配列で構成されたDNA又はRNAであり、
前記X、Y、及びZのいずれか1つがDNAであるとき、他の2つの1つ以上は、RNAであることを特徴とする、スモールRNA(small RNA)を検出する方法。 - 前記Xは、1〜60個の塩基配列で構成されたDNA又はRNA、
前記Yは、1〜10個の塩基配列で構成されたDNA又はRNA、及び
前記Zは、0〜10個の塩基配列で構成されたDNA又はRNAであり、
前記Zが0であるとき、Yは、3〜10個の塩基配列で構成されたDNA又はRNAであり、
前記Zが1であるとき、Yは、2〜10個の塩基配列で構成されたDNA又はRNAで構成されたことを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。 - 前記検出可能なマーカーは、前記一本鎖核酸に共有結合又は非共有結合により結合する蛍光物質、又は蛍光物質と消光物質で構成された蛍光ペアであることを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。
- 前記一本鎖核酸(promer)のX、Y、及びZは、非特異的な切断を防ぐために全体的にメチル化されるか、又は部分的にメチル化されるように合成されたことを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。
- 前記一本鎖核酸(promer)のXは、10〜30個の塩基配列で構成されたDNA又はRNAであり、Yは、1〜10個の塩基配列で構成されたDNA又はRNAであり、前記Zは、2〜10個の塩基配列で構成されたDNA又はRNAであることを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。
- 前記酵素は、Y部位がDNAであるとき、DNAヌクレアーゼ(DNA nuclea
se、DNase)、具体的には、DNaseII、DNaseII、S1ヌクレアーゼ、ヌクレアーゼP1、APエンドヌクレアーゼ、UvrABCヌクレアーゼを用い、X部位がRNAであるとき、RNA加水分解酵素(ribonuclease、RNase)、具体的には、RNaseII、RNaseIII、RNaseIV、RNaseH、又はRNase T2を用いることを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。 - 前記c)工程又はc−1)工程は、前記b)工程又はb−1)工程で合成されたcDNAと結合した前記一本鎖核酸が、前記一本鎖核酸のY部位を切断する酵素により前記一本鎖核酸のY部位が切断され、Y及びZ部位が分離され、X部位がプライマーとして機能して増幅されることを特徴する、請求項1に記載のスモールRNA(small RNA)を検出する方法。
- 前記c−1)工程は、(i)前記一本鎖核酸がフォワードプライマー及びプローブとして使用される場合、cDNAの一部の塩基配列と相補的結合が可能な塩基配列で構成されたリバースプライマーがさらに含まれ、(ii)前記一本鎖核酸がリバースプライマー及びプローブとして使用される場合、cDNAの一部の塩基配列と相補的結合が可能な塩基配列で構成されたフォワードプライマーがさらに含まれることを特徴とする、請求項1に記載のスモールRNA(small RNA)を検出する方法。
- a)X−Y−Zの構造を有する前記一本鎖核酸と相補的な塩基配列を有する核酸が付着している抗体を調製する工程と、b)生物学的試料から得られたスモールRNAに関連するタンパク質と、前記a)工程で調製した抗体とを結合させて、タンパク質−抗体複合体を形成させる工程と、c)前記複合体にX−Y−Zの構造を有する前記一本鎖核酸をハイブリダイズさせ、タンパク質−抗体−一本鎖核酸複合体を形成させる工程と、d)前記タンパク質−抗体−一本鎖核酸複合体を切断試薬で処理して、一本鎖核酸断片を抗体から分離させた後に、分離された前記一本鎖核酸断片の量を測定して、スモールRNA(small RNA)に関連するタンパク質を検出する工程と、を含むスモールRNA(small RNA)に関連するタンパク質分子を検出する方法であって、
前記一本鎖核酸は、X−Y−Zの構造を有し、前記一本鎖核酸の両末端又は内部に1つ又はそれ以上の検出可能なマーカーが付されており、リアルタイム検出を目的とする標的核酸又は標的タンパク質の特定部位に結合して複合体を形成し、特定の酵素によりY部位の切断時、X部位は、標的核酸又は標的タンパク質と複合体を維持しながらプライマーとして作用し、Y及びZは、前記標的核酸又は標的タンパク質の特定の部位から分離されることを特徴とし、
前記X、Y、及びZは、塩基配列で構成されたDNA又はRNAであり、
前記X、Y、及びZのいずれか1つがDNAであるとき、他の2つの1つ以上は、RNAであることを特徴とする、スモールRNA(small RNA)に関連するタンパク質分子を検出する方法。 - 前記d)工程は、前記a)工程で調製した抗体と結合した前記一本鎖核酸が、Y部位を切断する酵素により前記一本鎖核酸のY部位が切断され、Y及びZ部位が分離され、X部位がプライマーとして機能して増幅されることを特徴とする、請求項9に記載のスモールRNA(small RNA)に関連するタンパク質分子を検出する方法。
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KR20210109745A (ko) * | 2020-02-28 | 2021-09-07 | 주식회사 누리바이오 | 단일 표적 유전자의 유전적 변이 실시간 검출용 단일핵산 및 이를 이용한 검출 방법 |
CN113624980B (zh) * | 2021-08-09 | 2023-06-13 | 四川大学华西医院 | 基于识别诱导的恒温扩增技术对蛋白质进行检测的方法及试剂盒 |
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WO2005052127A2 (en) * | 2003-11-25 | 2005-06-09 | Myun Ki Han | Real-time detection of nucleic acids and proteins |
JP2010516284A (ja) * | 2007-01-26 | 2010-05-20 | ストラタジーン カリフォルニア | マイクロrnaの検出のための方法、組成物及びキット |
US20130310269A1 (en) * | 2012-05-04 | 2013-11-21 | Austin So | Hot-start digital pcr |
JP2016510601A (ja) * | 2013-03-15 | 2016-04-11 | インテグレイテツド・デイー・エヌ・エイ・テクノロジーズ・インコーポレイテツド | 修飾RNAモノマーを用いたRNaseH系アッセイ |
US20160265031A1 (en) * | 2015-03-13 | 2016-09-15 | Life Technologies Corporation | Methods, compositions and kits for small rna capture, detection and quantification |
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CA2209080C (en) | 1994-12-30 | 2008-07-29 | Georgetown University | Fluorometric assay for detecting nucleic acid cleavage |
US8618253B2 (en) | 2010-05-25 | 2013-12-31 | Samsung Techwin Co., Ltd. | Modified RNAse H and detection of nucleic acid amplification |
KR101657464B1 (ko) | 2014-08-05 | 2016-09-20 | 주식회사 포스코 | 철계 분말의 제조방법 |
KR102554638B1 (ko) | 2015-08-14 | 2023-07-12 | 한국전자통신연구원 | 면허 및 비면허 대역들을 지원하는 네트워크에서 통신 노드의 동작 방법 |
KR101901749B1 (ko) * | 2016-02-15 | 2018-09-28 | 주식회사 누리바이오 | 실시간 핵산 또는 단백질 검출용 단일핵산 및 이를 이용한 검출 방법 |
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- 2017-09-27 CN CN201780059846.4A patent/CN110023507B/zh active Active
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Patent Citations (5)
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WO2005052127A2 (en) * | 2003-11-25 | 2005-06-09 | Myun Ki Han | Real-time detection of nucleic acids and proteins |
JP2010516284A (ja) * | 2007-01-26 | 2010-05-20 | ストラタジーン カリフォルニア | マイクロrnaの検出のための方法、組成物及びキット |
US20130310269A1 (en) * | 2012-05-04 | 2013-11-21 | Austin So | Hot-start digital pcr |
JP2016510601A (ja) * | 2013-03-15 | 2016-04-11 | インテグレイテツド・デイー・エヌ・エイ・テクノロジーズ・インコーポレイテツド | 修飾RNAモノマーを用いたRNaseH系アッセイ |
US20160265031A1 (en) * | 2015-03-13 | 2016-09-15 | Life Technologies Corporation | Methods, compositions and kits for small rna capture, detection and quantification |
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JP2022185060A (ja) | 2022-12-13 |
CN110023507B (zh) | 2024-01-23 |
US20190226033A1 (en) | 2019-07-25 |
WO2018062859A1 (ko) | 2018-04-05 |
KR101991440B1 (ko) | 2019-06-24 |
EP3521450A1 (en) | 2019-08-07 |
KR20180055759A (ko) | 2018-05-25 |
EP3521450A4 (en) | 2020-04-08 |
BR112019005790A2 (pt) | 2019-06-18 |
CN110023507A (zh) | 2019-07-16 |
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