JP2016501028A - 固相デバイスにおける核酸の単離及び分析のためのラベルフリー方法 - Google Patents
固相デバイスにおける核酸の単離及び分析のためのラベルフリー方法 Download PDFInfo
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Abstract
Description
methylation: Molecular mechanisms and clinical implications. Clin. Cancer Res. 15 (2009) 3927−3937.; M. Wielscher, et al. Methyl−binding domain protein−based DNA isolation from human blood serum combines DNA analyses and serum−autoantibody testing. BMC Clin. Pathol. 11 (2011) 11−20.; and B.R. Cipriany, et al. Real−time analysis and selection of methylated DNA by fluorescence−activated single molecule sorting in a nanofluidic channel. Proc. Nat. Acad. Sci USA. 109 (2012) 8477−8482.)。ヒストンアセチラーゼ,ヒストンメチルトランスフェラーゼ,及びATP依存性クロマチンリモデリング酵素との相互作用を介して、MBDは、メチル化DNAを、転写に関して抑圧的なコンパクトなクロマチン環境へ変換する。特に、MBDは、MeCP2タンパク質のメチルCpG結合ドメインである。MeCP2タンパク質は、いずれのシーケンス前後関係においてもメチル化CpGを対称的に結合し、メチル化依存性転写抑制を媒介することに関わっている。MeCP2は、もっぱらin vivoでメチル化DNAフラグメントを結合するという強力な証拠があるにも関わらず、in vitroにおけるMeCP2のDNAメチル化依存性結合活性は、一般のin vitroDNA分析に好適なものにする、一致した文献内にも記載された[S.B. Baylin; McCabe, et al.; M. Wielscher, et al.;及びB.R. Cipriany, et al.]。
(i)核酸とDMAとの複合体(complex)を形成させる条件で、アジプイミド酸ジメチル(DMA)とともに核酸サンプルをインキュベートするステップと;
(ii)ステップ(i)の複合体を、固相デバイスの表面と接触させるステップと;
(iii)前記複合体の核酸を単離及び/又は分析するステップと
を有する、固相デバイスにおける核酸分子の単離及び/又は分析のための方法を包含する。
(i)核酸分子に直接結合可能なアジプイミド酸ジメチル(DMA)化合物;及び
(ii)核酸とDMAとの相互作用のための固体(solid)表面
を有する核酸サンプルにおける興味対象の核酸分子を単離するシステムを包含する。
(i)核酸とDMAとの複合体を形成させる条件で、アジプイミド酸ジメチル(DMA)とともに核酸サンプルをインキュベートするステップと;
(ii)ステップ(i)の複合体を、固相デバイスの表面と接触させるステップと;
(iii)前記複合体の核酸を単離及び/又は分析するステップと
を有する、固相デバイスにおける核酸分子の単離及び/又は分析のための方法を包含する。
(i)光学検出システムの検出器により測定された出力光強度を決定するステップと;
(ii)インキュベーションの間、前記光学検出システムの共振器の有効屈折率における変化を決定するステップと;
をさらに有してよい。
(i)核酸分子に直接結合可能なアジプイミド酸ジメチル(DMA)化合物;及び
(ii)核酸とDMAとの相互作用のための固体表面
を有する核酸サンプルにおける興味対象の核酸分子を単離するためのシステムを包含する。
いくつかの実施形態において光源から出力された光の一部は、例えば光源の発光波長を決定するため、サンプルされる。
(1)細胞サイズに基づき細胞を濾過及び分離するステップ、
(2)細胞を溶解するステップ、
(3)DNAと、カオトロピック試薬又はDMAのいずれかと結合するステップ、
(4)DNAを洗浄及び精製するステップ、及び
(5)DNAを、蒸留水又は溶出緩衝液のいずれかで溶出するステップ。
抽出DNAの溶出後、3種の異なるテクニックにより抽出されたDNAの純度を、260nm(DNA)及び280nm(タンパク質)におけるサンプルの光学密度の比率を決定することにより測定した。血液及び尿両方におけるDMAテクニック由来のDNAの質は、エタノールベースの方法から得られたものと比べて、サンプルについて統計的に顕著な誘導された純度であった(p<0.001)。事実、尿媒体は比較的正常であり、血液と比べてタンパク質をほとんど含まないので、他の方法について血液サンプルから抽出されたDNAの品質に対するDMAベースのSPEの有効性は、尿サンプル由来のものよりも明白であろうことが期待された。予想とは反対に、提案されたテクニックにより高度に生成されたDNAが、全血及び尿サンプル両方から得られた。これは、カオトロピック方法と比べて、他の分子(例えば細胞破片、タンパク質、その他)を取り除くための洗浄ステップの際、DMAが、液体サンプルから抽出されたDNA分子と緊密に結合しているからだと思われる。次に、RT−PCRを使用する抽出DNAで、増幅テストを実施した。HRAS及びアクチン遺伝子を、遺伝子ターゲットとして使用し、3種の異なるテクニックを使用することにより抽出された全DNAサンプルにおいて増幅した。DMAベースのテクニックで抽出されたDNAの量及び質は、そのほかの方法で得られたものよりも大きかったことが示された。リアルタイム(RT)−PCRのため、合計DNAサンプルグループから抽出されたDNA2μLを、いずれの定量化なしに、前分析システムのための実際の状況を真似るために使用した。当該システムにおいて、デバイスから抽出されたDNAは、臨床現場において遺伝子分析のいずれの定量化なしに、ターゲットテンプレートとして直接使用されることが期待される。従って、QIAmp DNAミニキットを使用するSYBR蛍光信号(全血又は尿200μLから抽出されたDNA)は、他より速いCt(サイクル閾値)値において飽和されているようである。DMAベースのテクニックを使用することにより、遺伝子分析のためのDNAバイオマーカー(HRAS,アクチン)は良い品質及び量で増幅されたことが観察された。従って、本研究において提案されたDMAテクニックは、マイクロチップシステムにおけるヒト体液の小体積由来の高品質を有する固相DNA抽出のために有用であり得る。
マイクロチップ環境においてDMAベースの方法を試験するため、ケイ素系DNA抽出マイクロ流体デバイスを使用した。DNA抽出マイクロ流体デバイスの構造及び制帽は知られている。ケイ素マイクロ流体チップは、3つの成分を有する:
(1)細胞分離のためのプレ濾過部分;
(2)細胞溶解のための2ステージスパイラルミキサーからなるマイクロミキサー;及び
(3)SiO2表面積の最大化のため(60mm2を超えると推定される)、DMAベースの方法のための曲がりくねった形状のマイクロチャネル。
マイクロ流体チップは、硬質マスキング層として2μm厚さの熱SiO2が積層されたケイ素基板の正面側に、反応性イオンエッチング(RIE)プロセスを使用して製造された。流体相互接続を形成するため、水酸化カリウム(KOH)によるウェットエッチプロセスを、熱SiO2層の上、低圧化学蒸着(LPCVD)により堆積された窒化ケイ素(Si3N4)のコンポジットマスキング層が積層されたケイ素基板の裏側に適用した。マイクロチャネル表面上の残存するSiO2薄膜層と共に、開いたチャネルをキャップするため、Pyrex(登録商標)ガラス基板とのアノード結合プロセスを実施した。各製造マイクロ流体デバイスの全体サイズは、16mm×12mm×1.2mmである。
入口I:分離−サイズによって細胞を分離するため、サンプルをマイクロフィルタ内へ注入する;
入口II:分泌―溶解緩衝液として使用されるべきプロテイナーゼKを有するAL緩衝液を、マイクロミキサー内に注入する;
入口III:洗浄及び溶出−サンプルを精製するため、洗浄緩衝液(EtOHもしくはDMAのいずれか及びPBS)を注入する;及び
出口IV:ケイ素固体表面から核酸を溶出する。
カオトロピック方法(エタノール)を使用する場合、8μLのPBS緩衝液を有する全血(2μL)又は尿(10μL)のいずれかが、入口I内へ、流速1.67μL分−1で10分間、シリンジポンプ(KDScientific,MA)で注入された。次いで、溶解緩衝液が入口II内へ、流速3μL分−1で10分間、シリンジポンプによるPBS緩衝液と共に3μL分−1で10分間までの入口I流速における増加と共に、注入された。溶解反応の際、エタノールを流速10μL分−1で10分間、入口IIIに添加した。次いで、エタノールのみを12.5μL分−1で5分間、チップ全体を洗浄するため、順次使用した。最後に、抽出DNAを、12.5μL分−1で10分間、純水で溶出した。
Claims (21)
- (i)核酸とDMAとの複合体を形成させる条件で、アジプイミド酸ジメチル(DMA)とともに核酸サンプルをインキュベートするステップと;
(ii)ステップ(i)の複合体を、固相デバイスの表面と接触させるステップと;
(iii)前記複合体の核酸を単離及び/又は分析するステップと
を有する、固相デバイスにおける核酸分子の単離及び/又は分析のための方法。 - インキュベーションの前に、固相デバイスの表面は官能化している、請求項1に記載の方法。
- 核酸サンプルは、プロテアーゼ、好ましくはプロテイナーゼKで抽出される、請求項1又は2に記載の方法。
- 核酸はメチル化DNAを有する、請求項1乃至3のいずれか一項に記載の方法。
- 固相デバイスはマイクロ流体デバイスである、請求項1乃至4のいずれか一項に記載の方法。
- 固相デバイスはリング共振器である、請求項1乃至4のいずれか一項に記載の方法。
- (i)光学検出システムの検出器により測定された出力光強度を決定するステップと;
(ii)インキュベーションの間、前記光学検出システムの共振器の有効屈折率における変化を決定するステップと;
をさらに有する、請求項6に記載の方法。 - 複合体を溶出するステップをさらに有する、請求項1乃至7のいずれか一項に記載の方法。
- 前記リング共振器は導波管構造を有する、請求項6に記載の方法。
- 複合体は、非メチル化シトシン残基において変性される、請求項1乃至9のいずれか一項に記載の方法。
- 複合体もしくは変性複合体は増幅される、請求項1乃至10のいずれか一項に記載の方法。
- 複合体もしくは変性複合体は検出される、請求項1乃至11のいずれか一項に記載の方法。
- (i)核酸分子に直接結合可能なアジプイミド酸ジメチル(DMA)化合物;及び
(ii)核酸とDMAとの相互作用のための固体表面
を有する核酸サンプルにおける興味対象の核酸分子を単離するシステム。 - 固体表面は官能化される、請求項13に記載のシステム。
- 固体表面はマイクロ流体デバイスの上にある、請求項13又は14に記載のシステム。
- 固体表面はリング共振器の上にある、請求項13又は14に記載のシステム。
- リング共振器は、光学検出センサであって、前記核酸分子が前記DMAに結合される場合、前記検出センサは改変読取を有する、それにより核酸分子とDMAとの間に形成された複合体を感知するように、前記センサは構成される、請求項16に記載のシステム。
- 共振波長において共振するように、光学検出センサは構成される、請求項17に記載のシステム。
- 光学センサのための前記共振波長において光を提供可能な波長可変レーザをさらに有する、請求項18に記載のシステム。
- 光学センサもしくはリング共振器は導波管構造を有する、請求項17乃至19のいずれか一項に記載のシステム。
- 前記核酸分子が前記DMAに結合され、前記複合体を形成する場合、前記共振器は共振波長を有する、請求項17乃至20のいずれか一項に記載のシステム。
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US11584924B2 (en) | 2016-08-24 | 2023-02-21 | Infusion Tech | Method for concentrating microorganism or extracting nucleic acid using DTBP |
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JP6396911B2 (ja) | 2012-10-15 | 2018-09-26 | ナノセレクト バイオメディカル, インコーポレイテッド | 粒子を選別するためのシステム、装置、および、方法 |
WO2017200249A1 (ko) * | 2016-05-17 | 2017-11-23 | 울산대학교 산학협력단 | 고형상 대상물을 이용한 핵산 추출방법 |
KR101913208B1 (ko) | 2016-05-17 | 2018-10-30 | 울산대학교 산학협력단 | 고형상 대상물을 이용한 핵산 추출방법 |
WO2018038549A1 (ko) * | 2016-08-24 | 2018-03-01 | 울산대학교 산학협력단 | Dtbp를 이용한 미생물 농축 또는 핵산 추출 방법 |
WO2018169307A1 (ko) * | 2017-03-15 | 2018-09-20 | 울산대학교 산학협력단 | 규조토를 이용한 미생물 농축 방법 및 핵산 추출 방법 |
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WO2019078638A2 (ko) * | 2017-10-18 | 2019-04-25 | 울산대학교 산학협력단 | 동형2기능성 이미도에스터를 이용한 병원체 농축 방법 |
WO2019088527A2 (ko) * | 2017-10-30 | 2019-05-09 | 울산대학교 산학협력단 | 산화아연 나노스타를 이용한 병원체 용균 및 핵산 추출 방법 |
EP3705581A4 (en) | 2017-10-30 | 2021-08-18 | Infusion Tech | PROCESS FOR LYSIS OF PATHOGENS AND EXTRACTION OF NUCLEIC ACIDS USING ZINC OXIDE NANOSTARES |
WO2019088429A1 (ko) * | 2017-10-30 | 2019-05-09 | 울산대학교 산학협력단 | 동형2기능성 이미도에스터를 이용한 액체생검 유래 생체물질의 추출 방법 |
KR102260012B1 (ko) * | 2020-09-29 | 2021-06-03 | 주식회사 에이아이더뉴트리진 | 미생물 농축 또는 핵산 추출용 조성물 및 이를 이용한 미생물 농축 또는 핵산 추출 방법 |
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ES2706761T3 (es) | 2019-04-01 |
EP2931921B1 (en) | 2018-10-31 |
US9856520B2 (en) | 2018-01-02 |
WO2014092653A1 (en) | 2014-06-19 |
TR201820335T4 (tr) | 2019-01-21 |
CN104968803B (zh) | 2019-07-26 |
KR20150096444A (ko) | 2015-08-24 |
US20150322486A1 (en) | 2015-11-12 |
CN104968803A (zh) | 2015-10-07 |
SG11201504388XA (en) | 2015-07-30 |
EP2931921B8 (en) | 2019-01-09 |
US20180073061A1 (en) | 2018-03-15 |
KR102091041B1 (ko) | 2020-03-23 |
EP2931921A4 (en) | 2016-08-24 |
JP6455438B2 (ja) | 2019-01-23 |
EP2931921A1 (en) | 2015-10-21 |
US10584373B2 (en) | 2020-03-10 |
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