JP6158808B2 - 流体を処理するためのシステム及び方法 - Google Patents
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Description
全血から白血球核を精製するためのバンプアレイプロセス
単一のアレイによって図10に示す操作1002及び1004を実施する。これらはそれぞれ血漿からの細胞の分離及び白血球核の単離を含む(図5に示す)。斯かるアレイは、図5に模式的に示すものであり、直径が>3〜4μmの全粒子をバンプするように設計される。臨界寸法をこのサイズに設定することで、細胞(赤血球細胞(RBC)6〜8μm、白血球(WBC)6〜16μm)及び核(5〜9μm)が流通方向に対して斜めにバンプされる。血液はアレイ上の緩衝生理食塩水を満たした領域に誘導される。この領域にバンプされた細胞から、血漿が洗い落とされる。細胞は更にアレイに対して横方向に進行し、溶解緩衝液の流体流へと誘導される。溶解緩衝液は、RBC及びWBCを溶解させるが、WBCの核は溶解させないような濃度の非イオン性界面活性剤を含む。核は依然としてアレイの臨界直径(3〜4mm)よりも大きいので、細胞と同じく斜め方向の軌道を辿る。溶解緩衝液から放出された核は洗浄緩衝液(界面活性剤を含まない溶解緩衝液)の流体流に誘導され、アレイの右下側近傍の出側貯留部で回収される。
単離された白血球核からHMW DNAを精製するためのバンプアレイプロセス
次のプロセスは細胞核からのDNAの精製である。このシステムでは斯かる作業を実現するために、カオトロピック塩溶液(4Mチオシアン酸グアニジン、GuSCN)を用いて核を溶解し、染色体タンパク質をDNAから解離させている。カオトロープによる溶解は、一般的なSDS−プロテナーゼKプロトコールよりも早くて確実である。高濃度のカオトロープの存在下で、酸化ケイ素表面に結合するのを防止するため、シリコンアレイをフルオロシランによって科学的に被覆する。
NGSライブラリー形成のためのバンプアレイプロセス
実施例2のアレイ(図10に示す操作1006及び1008の例)により精製されたHMWゲノムDNAを、転位媒介ライブラリー形成反応(図7参照)を実施するアレイに導入する。斯かるライブラリーアレイのアレイ配置は、実施例2の第二のアレイ(図10に示す操作1008の例)と同様である。即ち、〜40kb以上のDNAが右にバンプされ、トランスポゼース系ライブラリー形成試薬流に導入される(図7参照)。
ヒト血液試料からの細菌DNAの単離のためのバンプアレイプロセス
工程1.RBC及びWBCが溶解されるがWBC核は無傷に維持される条件下で、ヒト血液の試料を非イオン性界面活性剤で処理する(0.32Mスクロース、5mM MgCl2、1% Triton X−100、0.01M Tris−HCl、pH7.6)。これらの条件は細菌を溶解させるほど強くはないが、細菌は溶解物中で無傷細胞形態として維持される。
Claims (22)
- 生体流体の処理のためのシステムであって、少なくとも1つの入側貯留部(input reservoir)と、少なくとも1つのバンプアレイ機構と、少なくとも1つの出側貯留部(output reservoir)と
を含み、
前記少なくとも1つの入側貯留部は、生体流体を受容して、前記生体流体から1又は2以上の細胞を分離するように構成され、
前記少なくとも1つのバンプアレイ機構は、複数の処理領域を含むように構成され、前記複数の処理領域は、第一の緩衝液を含む第一の処理領域と、少なくとも1つの配列決定アダプター(sequencing adaptor)を有するトランスポゼース複合体を含む第二の処理領域とを含み、
ここで前記第一の処理領域は、前記1又は2以上の細胞が前記第一の処理領域を通過する際に、前記1又は2以上の細胞から精製されたデオキシリボ核酸(deoxyribonucleic acid:DNA)を生成するように構成され、
前記第二の処理領域は、前記精製されたDNAを受容して、少なくとも1つの配列決定アダプターを有するトランスポゼース複合体と混合し、第二の溶液を調製するように構成され、
前記少なくとも1つの出側貯留部は、前記第二の溶液を受容するように構成される、システム。 - 生体流体が、全血、尿、脊髄液、唾液、口腔スワブ(buccal swabs)、痰、気管支洗浄液、胃洗浄液、微生物培養培地、糞、軟膜(buffy coat)、血清、血漿、血小板濃縮物、水試料、及び/又は他の任意の生物学的、化学的、及び/又は、生化学的流体、及び/又は、それらの任意の組み合わせのうち少なくとも1つを含む、請求項1に記載のシステム。
- 少なくとも1つのバンプアレイが、決定論的側方変位を用いて、前記精製されたDNA及び前記第二の溶液の少なくとも一方を処理する、請求項1に記載のシステム。
- 複数のバンプアレイの連鎖配列を用いて、前記精製されたDNA及び前記第二の溶液の少なくとも一方を処理することを更に含む、請求項1に記載のシステム。
- 生体流体が全血である、請求項1に記載のシステム。
- 前記少なくとも1つの出側貯留部が、流出溶液に含まれる少なくとも1つの細胞のサイズに基づいて、流出溶液を分画する、請求項1に記載のシステム。
- 前記入側貯留部を複数含む、請求項1に記載のシステム。
- 前記複数の入側貯留部が、少なくとも前記生体流体を有する第一の貯留部と、前記第一の緩衝液を有する第二の貯留部と、前記少なくとも1つの配列決定アダプターを有するトランスポゼース複合体を有する第三の貯留部とを含む、請求項1に記載のシステム。
- 各貯留部の内容物を前記少なくとも1つのバンプアレイに導入するように構成された複数の導入口を更に含む、請求項1に記載のシステム。
- 粒子トラップを更に含む、請求項1に記載のシステム。
- 前記少なくとも1つのバンプアレイが前記粒子トラップを含むように構成される、請求項10に記載のシステム。
- 前記第一の緩衝液が細胞溶解緩衝液を含む、請求項1に記載のシステム。
- 前記複数の処理領域が、洗浄緩衝液を含む第三の処理領域と、グアニジンイソチオシアネート(guanidine isothiocyanate:GuSCN)緩衝液を含む第四の処理領域とのうち少なくとも一方を更に含む、請求項1に記載のシステム。
- 前記洗浄緩衝液が核洗浄緩衝液を含む、請求項13に記載のシステム。
- 前記GuSCN緩衝液が、核を分離し、DNAから各タンパク質を除去するように構成される、請求項13に記載のシステム。
- 少なくとも1つの化学的及び/又は酵素試薬流を用い、少なくとも1つのバンプアレイを用いた、高分子量(high molecular weight:HMW)核酸の順次処理のための方法であって、ここでHMW核酸の有効流体力学半径は、少なくとも1つのバンプアレイの臨界サイズよりも大きく、ここで当該方法は、
・少なくとも1つのバンプアレイでHMW核酸を受容し、
・HMW核酸を少なくとも1つの化学的及び/又は酵素試薬流と接触させることを含み、
ここで少なくとも1つの化学的及び/又は酵素試薬流を、バンプアレイ内におけるHMW核酸フローの方向に流通させ、
ここでHMW核酸が、少なくとも1つの化学的及び/又は酵素試薬流と反応する、方法。 - 少なくとも1つの化学的及び/又は酵素試薬流を用い、少なくとも1つのバンプアレイを用いた、核酸の順次処理のための方法であって、当該方法は、
・核酸を受容し、
・核酸を少なくとも1つのバンプアレイに流通させ、
・少なくとも1つのバンプアレイを用いて、核酸を少なくとも1つの化学的及び/又は酵素試薬流と接触させ、
・少なくとも1つの化学的及び/又は酵素試薬流を用いて核酸を修飾し、
・少なくとも1つのバンプアレイを用いて、精製された核酸を、少なくとも1つの化学的及び/又は酵素試薬流から除去する
ことを含む、方法。 - 複数のバンプアレイが核酸を順次処理し、ここで複数のバンプアレイ内の個々のバンプアレイは直列に連結され、複数のバンプアレイ内の一のバンプアレイの出流が、それに続く複数のバンプアレイ内の別のバンプアレイの入流として供される、請求項17に記載の方法。
- 単一のバンプアレイで、流通、接触、修飾、及び除去を実施する、請求項17に記載の方法。
- 核酸が高分子量(HMW)核酸である、請求項17に記載の方法。
- 核酸がデオキシリボ核酸(DNA)であり、DNAはバンプアレイ内において、DNAを担持する少なくとも1つのマイクロ粒子に連結される、請求項17に記載の方法。
- DNAが共有結合及び非共有結合のうち少なくとも1つによって連結される、請求項21に記載の方法。
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US201161515063P | 2011-08-04 | 2011-08-04 | |
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PCT/US2012/049603 WO2013020089A2 (en) | 2011-08-04 | 2012-08-03 | Systems and methods for processing fluids |
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US10323279B2 (en) | 2012-08-14 | 2019-06-18 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10400280B2 (en) | 2012-08-14 | 2019-09-03 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10752949B2 (en) | 2012-08-14 | 2020-08-25 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
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US20150166986A1 (en) | 2015-06-18 |
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