JP6367196B2 - マイクロカプセル組成物および方法 - Google Patents
マイクロカプセル組成物および方法 Download PDFInfo
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Description
本願は、2012年8月14日に出願された米国特許仮出願第61/683,192号;2012年12月14日に出願された米国特許仮出願第61/737,374号;2013年2月8日に出願された米国特許仮出願第61/762,435号;2013年3月15日に出願された米国特許仮出願第61/800,223号;2013年6月27日に出願された米国特許仮出願第61/840,403号;および2013年7月10日に出願された米国特許仮出願第61/844,804号の利益を主張するものであり、これらの出願は、あらゆる目的で参照によりそれらの全体が本明細書に組み込まれる。
本明細書で言及されたあらゆる刊行物、特許および特許出願は、あらゆる目的で、さらに個々の刊行物、特許または特許出願のそれぞれが参照により本明細書に組み込まれることを明確かつ個別に示されたのと同程度に、参照によりそれらの全体が本明細書に組み込まれる。
本開示は、マイクロウェルアレイ装置または他の区分カプセルアレイ装置と、斯かる装置を用いる方法を提供する。一般に、本装置は、多くの場合、区分当たり特定のマイクロカプセル濃度でマイクロカプセルが充填された区分(例えば、マイクロウェル、液滴)の集合体である。
図1Aは、固体または半透性の殻または膜110によって封入された第2の層130に包まれた内部区画120を含む例示的なマイクロカプセルの模式図である。一般に、殻は、周囲の環境(例えば、マイクロウェルの内部)から内部区画を隔てている。内部区画、例えば、120、130は、試薬等の材料を含むことができる。図1Aに描写されている通り、試薬100は、内部区画120に存在することができる。しかし、一部の事例において、試薬は、エンベロープ層(enveloping layer)130にまたは両方の区画に位置する。一般に、マイクロカプセルは、特定のトリガーの導入後に、内部材料またはその一部を放出することができる。トリガーは、殻層110および/または内部エンベロープ層130の破壊を引き起こし、マイクロウェルの空洞等の外部環境と内部区画100、120との接触を可能にすることができる。
本開示の装置のマイクロカプセルは、多数の方法およびプロセスにより調製することができる。調製技法としては、パンコーティング、噴霧乾燥、遠心押し出し(centrifugal extrusion)、エマルションに基づく方法および/またはマイクロ流体技法が挙げられる。典型的には、調製のための方法は、マイクロカプセルの所望の特徴に基づき選ばれる。例えば、方法を選ぶ際に、殻壁の厚さ、透過性、殻壁の化学組成、殻壁の機械的統合性およびカプセルサイズを考慮に入れることができる。調製方法は、コア材料(例えば、流体、試薬等)が、水性、有機または無機であるか等、カプセル内に特異的な材料を取り込む能力に基づき選択することもできる。その上、調製方法は、マイクロカプセルの形状およびサイズに影響を与えることができる。例えば、カプセルの形状(例えば、球体、楕円体等)は、前駆液体における液滴の形状によって決まる可能性があり、これは、コア液体の粘性および表面張力、エマルションの流れの方向、液滴安定化において用いられた界面活性剤の選択、ならびに特定のサイズ(例えば、マイクロカプセルをマイクロチャネルまたはキャピラリー内に適合させるために、マイクロカプセルの歪みを要求するサイズ)のマイクロチャネルまたはキャピラリーにおいて作製された調製物等の物理的閉じ込めにより決定され得る。
マイクロカプセルは、広範な化学的特徴を有する種々の材料を含むことができる。一般に、マイクロカプセルは、所望の形状およびサイズであり、マイクロカプセル中で貯蔵しようとする試薬と適合性のマイクロカプセルを形成する能力を有する材料を含む。
マイクロカプセルは、多数のサイズまたは形状のいずれであってもよい。一部の事例において、マイクロカプセルの形状は、球体、楕円体、円柱状、六角形または他のいずれかの対称的もしくは非対称的形状であってもよい。マイクロカプセルのいずれかの断面も、いかなる適切な形状のものであってもよく、その例としては、これらに限定されないが、円形、長円形、正方形、長方形、六角形または他の対称的もしくは非対称的形状が挙げられる。一部の事例において、マイクロカプセルは、装置の開口部(例えば、マイクロウェルの表面)を補完する特異的な形状のものであってよい。例えば、マイクロカプセルが球体であれば、装置のマイクロウェルの開口部も円形であってもよい。
本明細書に提示されている装置は、遊離の試薬および/またはマイクロカプセルに封入された試薬を含むことができる。試薬は、分析物の試料調製反応に適した種々の分子、化学物質、粒子および要素であってもよい。例えば、標的のDNA配列決定のための試料調製反応において用いられるマイクロカプセルは、次の試薬:酵素、制限酵素[例えば、多重カッター(multiple cutter)]、リガーゼ、ポリメラーゼ(例えば、dUTPおよび/またはウラシルを認識するおよび認識しないポリメラーゼ)、フルオロフォア、オリゴヌクレオチドバーコード、バッファー、デオキシヌクレオチド三リン酸(dNTP)[例えば、デオキシアデノシン三リン酸(dATP)、デオキシシチジン三リン酸(dCTP)、デオキシグアノシン三リン酸(dGTP)、デオキシチミジン三リン酸(dTTP)、デオキシウリジン三リン酸(dUTP)]、デオキシヌクレオチド三リン酸(ddNTP)その他。別の例において、単一細胞解析のための試料調製反応において用いられるマイクロカプセルは、次の試薬のうち1種または複数等の試薬を含むことができる:溶解バッファー、洗剤、フルオロフォア、オリゴヌクレオチドバーコード、リガーゼ、プロテアーゼ、熱活性化可能プロテアーゼ、プロテアーゼまたはヌクレアーゼ阻害剤、バッファー、酵素、抗体、ナノ粒子その他のうち1種または複数を含むことができる。
試料調製の後または最中に、個々の分子または分析物を同定および追跡する選択肢を保持することが望ましくなり得る。一部の事例において、本技術分野において「分子バーコード」として知られることもある、1種または複数の固有の分子識別子は、試料調製試薬として用いられる。このような分子は、オリゴヌクレオチドバーコード、抗体もしくは抗体断片、フルオロフォア、ナノ粒子および他の要素またはこれらの組み合わせ等の種々の異なる形態を含むことができる。特異的な適用に応じて、分子バーコードは、標的分析物に可逆的にまたは不可逆的に結合して、試料調製後に装置から回収した後に、個々の分析物の同定および/または定量化を可能にすることができる。
調製後に、試薬を充填したマイクロカプセルを、種々の方法を用いて装置中に充填することができる。マイクロカプセルは、一部の事例において、「乾燥カプセル」として充填することができる。調製後に、カプセルは、これらに限定されないが、分画遠心分離、液体相の蒸発、クロマトグラフィー、濾過その他等の様々な技法を用いて液体相から分離することができる。「乾燥カプセル」を粉末または粒子状物質として収集し、次に、マイクロウェルアレイのマイクロウェル中に置くことができる。空のウェルおよびマイクロウェルアレイにわたるマイクロカプセルの不十分な分布等の「湿潤カプセル」の充填が充填を無効にする事例において、「乾燥カプセル」の充填が、好ましい方法となり得る。
/cm3、2.3g/cm3、2.4g/cm3または2.5g/cm3であってもよい。斯かる密度は、いずれか特定の流体(例えば、水性物、水、油等)中におけるマイクロカプセルの密度を反映することができる。
A.構造/特色
本開示の装置は、複数の孔、空洞を含有する固体プレートを含むマイクロウェルアレイであってもよく、またはマイクロカプセルおよび/もしくは分析物が置かれたマイクロウェルであってもよい。一般に、流体試料(または分析物)は、装置中に導入され(例えば、入口を通して)、次に試料を複数のマイクロウェルへと分布させる流路を通って移動する。一部の事例において、追加の流体は、同様に装置中に導入される。試料が導入される際に、または一部の事例において、試料の導入後にマイクロカプセルがマイクロウェル中に導入される際に、マイクロウェルは、マイクロカプセルを含むことができる。
マイクロウェルアレイは、アルコール等の水性、非水性、油および有機溶媒を含む多数の異なる流体のいずれかを含むことができる。一部の事例において、流体を用いて、構成成分、例えば、試薬、マイクロカプセルまたは分析物を、マイクロウェル、アウトプットポート等の標的位置へと運搬する。他の事例において、流体を用いてシステムを流す。さらにまた他の事例において、流体を用いて、マイクロウェルを密封することができる。
分析物、遊離の試薬および/またはマイクロカプセルが装置に充填され、マイクロウェルに分布された後に、密封流体を用いて、マイクロウェル内でこれらをさらに区分化または単離することができる。密封流体を用いて、個々のウェルを密封することもできる。分析物、試薬および/またはマイクロカプセルの導入に用いた同じ入口ポートを通して密封流体を導入することができる。しかし、一部の事例において、密封流体は、別々の入口ポートにより、または複数の別々の入口ポートを通して装置に導入される。
本明細書に記載されている通り、分析物、遊離の試薬および/またはマイクロカプセルは、いずれかの適切な様式または順序で本装置に充填することができる。充填は、ランダムまたは非ランダムであってもよい。一部の事例において、正確な数の分析物および/またはマイクロカプセルが、個々のマイクロウェルそれぞれに充填される。一部の事例において、正確な数の分析物および/またはマイクロカプセルが、プレートにおけるマイクロウェルの特定のサブセットに充填される。さらにまた他の事例において、平均数の分析物および/またはマイクロカプセルが、個々のマイクロウェルそれぞれに充填される。さらに、本明細書に記載されている通り、一部の事例において、「乾燥」マイクロカプセルが、装置に充填されるが、他の事例において、「湿潤」マイクロカプセルが、装置に充填される。一部の事例において、「乾燥」および「湿潤」マイクロカプセル、および/または試薬の組み合わせは、同時にまたは経時的に装置に充填される。
様々な異なる刺激を用いて、マイクロカプセルまたはその内部区画からの試薬の放出を引き起こすことができる。一部の事例において、マイクロカプセルは分解性である。一般に、トリガーは、マイクロカプセルを包む殻もしくは膜の破壊もしくは分解、マイクロカプセルの内部の破壊もしくは分解、および/またはマイクロカプセルに試薬を固定化するいずれかの化学結合の破壊もしくは分解を引き起こすことができる。例示的なトリガーとしては、これらに限定されないが、化学的トリガー、バルクの変化、生物学的トリガー、光トリガー、熱的トリガー、磁気トリガーおよびこれらのいずれかの組み合わせが挙げられる。例えば、Esser-Kahnら(2011)Macromolecules 44:5539〜5553;Wangら(2009)ChemPhysChem 10:2405〜2409を参照されたい。
多数の化学的トリガーを用いて、マイクロカプセルの破壊または分解を引き起こすことができる。このような化学的変化の例としては、これらに限定されないが、殻壁へのpH媒介性の変化、架橋結合の化学的切断による殻壁の崩壊、引き起こされた殻壁の脱重合および殻壁のスイッチング反応が挙げられる。体積変化を用いて、マイクロカプセルの破壊を引き起こすこともできる。
生物学的刺激を用いて、マイクロカプセルの破壊または分解を引き起こすこともできる。一般に、生物学的トリガーは、化学的トリガーに似ているが、その多くの例は、酵素、ペプチド、糖類、脂肪酸、核酸その他等の生体分子または通常生物系に存在する分子を用いる。例えば、マイクロカプセルは、特異的プロテアーゼによる切断に対し感受性のペプチド架橋を有するポリマーを含むことができる。より具体的には、一例として、GFLGKペプチド架橋を含むマイクロカプセルを含むことができる。プロテアーゼカテプシンB等の生物学的トリガーの添加により、殻壁のペプチド架橋が切断され、カプセルの内容物が放出される。他の事例において、プロテアーゼを熱活性化してもよい。別の例において、マイクロカプセルは、セルロースを含む殻壁を含む。加水分解酵素キトサンの添加は、セルロース結合の切断、殻壁の脱重合およびその内部の内容物の放出のための生物学的トリガーとして役立つ。
マイクロカプセルは、熱的刺激を与えることによりその内容物を放出するよう誘導することもできる。温度の変化は、マイクロカプセルに種々の変化をもたらすことができる。熱の変化は、殻壁が崩壊するように、マイクロカプセルの融解をもたらすことができる。他の事例において、熱は、カプセルが破裂または爆発するように、カプセルの内部構成成分の内圧を増加させることができる。さらにまた他の事例において、熱は、カプセルを収縮した脱水状態へと変換させることができる。熱は、マイクロカプセルの殻内の感熱性ポリマーに作用して、マイクロカプセルを破壊させることもできる。
マイクロカプセルの殻壁への磁性ナノ粒子の包接は、カプセル破裂を引き起こすことに加えて、アレイにおいて粒子をガイドすることができる。本開示の装置は、あらゆる目的のための磁性粒子を含むことができる。一例において、カプセルを含有する多価電解質へのFe3O4ナノ粒子の取り込みは、振動磁場刺激の存在下で破裂を引き起こす。
マイクロカプセルは、電気刺激の結果として破壊または分解することができる。前の章に記載した磁性粒子と同様に、電気的感受性の粒子は、カプセルの破裂を引き起こすことに加えて、電場における整列化、導電率または酸化還元反応等の他の機能の両方を可能にすることができる。一例において、電気的感受性材料を含有するマイクロカプセルは、内部試薬の放出を制御できるように、電場において整列される。他の例において、電場は、殻壁それ自体の内の酸化還元反応を誘導することができ、これは、多孔度を増加させ得る。
本開示の装置は、斯かるトリガーまたは刺激をもたらすいずれかの器具または装置と組み合わせて用いることができる。例えば、刺激が熱である場合、本装置は、マイクロウェルの加熱を可能にし、カプセルの破裂を誘導することができる加熱または熱的制御プレートと組み合わせて用いることができる。これらに限定されないが、放射熱伝達、対流熱伝達または伝導熱伝達による加熱等の多数の熱伝達のいずれを熱刺激に用いてもよい。他の事例において、刺激が生物学的酵素である場合、各マイクロウェル内に置かれるように、酵素を装置中に注入することができる。別の一態様において、刺激が磁場または電場である場合、本装置は、磁気または電気プレートと組み合わせて用いることができる。
刺激を与え、カプセルを破裂させ、試薬を放出させた後に、装置内で試料調製反応を進行させることができる。試料反応において用いた試薬に応じて様々な期間、装置内で反応物をインキュベートすることができる。本装置は、試料調製反応に役立つ他の装置と組み合わせて用いることもできる。例えば、PCR増幅が望まれる場合、本装置は、PCRサーモサイクラーと組み合わせて用いることができる。一部の事例において、サーモサイクラーは、複数のウェルを含むことができる。区分が液滴である場合、液滴をサーモサイクラーのウェルに進入させることができる。一部の事例において、熱サイクルが開始される際に各ウェルにおいて複数の液滴が熱サイクル処理されるように、各ウェルは、複数の液滴を含むことができる。別の例において、反応が撹拌を必要とする場合、本装置は、振盪器具と組み合わせて用いることができる。
図4Aは、本開示の方法の多くの概略フローを提示し、図4Bは、図4Aの概略的注釈付きバージョンを提示する。試薬410を含有する1種または複数のマイクロカプセルは、マイクロウェルに予め充填され、続いて、この特定の図面においては核酸分析物420である分析物を添加することができる。次に、密封流体の適用等のいずれかの方法によってマイクロウェルを密封430することができる。入口および出口ポートは、例えば、蒸発を防止するために密封することもできる。これらの工程の後に、マイクロカプセル460を破壊し、マイクロウェルの内部への試薬450の放出を引き起こすために、刺激(例えば、熱、化学的、生物学的等)をマイクロウェルに加えることができる。その後、試薬が、細胞の溶解、タンパク質の消化、高分子量核酸の断片化またはオリゴヌクレオチドバーコードのライゲーション等の特定の機能を実行できるように、インキュベーション工程440を行うことができる。インキュベーション工程(必要に応じて行われる)に続いて、マイクロウェルの内容物を単独で、またはまとめて回収することができる。
本開示の装置は、分析物の操作、調製、同定および/または定量化において多種多様な用途を有することができる。一部の事例において、分析物は、細胞または細胞の集団である。細胞の集団は、均質(例えば、同じ細胞型の細胞株に由来、同じ種類の組織に由来、同じ器官に由来等)であってもよく、異質(異なる種類の細胞の混合物)であってもよい。細胞は、初代細胞、細胞株、組換え細胞、初代細胞、封入された細胞、遊離の細胞等であってもよい。
本明細書に開示されている装置は、分析物への固有の識別子または分子バーコードの割り当てを含む適用に用いることができる。多くの場合、固有の識別子は、分析物のタグ付けに用いられるバーコードオリゴヌクレオチドであるが、一部の事例において、異なる固有の識別子が用いられる。例えば、一部の事例において、固有の識別子は抗体であり、この場合、付着は、抗体および分析物の間(例えば、抗体および細胞、抗体およびタンパク質、抗体および核酸)の結合反応を含むことができる。他の事例において、固有の識別子は色素であり、この場合、付着は、分析物分子への色素のインターカレーション(DNAまたはRNAへのインターカレーション等)または色素で標識されたプローブへの結合を含むことができる。さらにまた他の事例において、固有の識別子は、核酸プローブとなることができ、この場合、分析物への付着は、核酸および分析物の間のハイブリダイゼーション反応を含むことができる。一部の事例において、反応は、識別子および分析物の間の化学結合を含むことができる。他の事例において、反応は、分析物への直接的な、または同位体で標識されたプローブによる、金属同位体の添加を含むことができる。
核酸配列決定は、特定の密度(例えば、マイクロウェル当たり約1個の分析物または本明細書に記載されている他の密度)におけるマイクロウェルへの試料分析物の物理的区分化から始まることができる。分析物が後に一体にプールされ、一斉に処理される場合であっても、核酸バーコードが個々の分析物に割り当てられると、その後の増幅および/または配列決定工程等のその後の工程において個々の分子を追跡することが可能になる。
本明細書に提示されている装置を用いて、位相またはリンケージ情報をその後に得ることができるような様式で分析物(例えば、核酸分析物)を調製することができる。斯かる情報は、長く伸びた核酸によって離間した遺伝的変種(例えば、SNP、突然変異、インデル、コピー数変種、トランスバージョン、転位置、逆位等)を含む、配列中の関連する遺伝的変異の検出を可能にすることができる。これらの変異は、シスまたはトランスいずれかの関係性で存在し得る。シス関係性において、2個以上の遺伝的変異は、同じポリ核酸分子または鎖に存在し得る。トランス関係性において、2個以上の遺伝的変異は、複数の核酸分子または鎖に存在し得る。
本明細書に提示されている装置を用いて、細胞特異的情報をその後に得ることができるような様式で、細胞分析物を調製することができる。斯かる情報は、細胞毎の遺伝的変異(例えば、SNP、突然変異、インデル、コピー数の変化、トランスバージョン、転位置、逆位等)の検出を可能にし、これにより遺伝的変異が同じ細胞に存在するか2個の異なる細胞に存在するかの決定を可能にすることができる。
「多重占有率(fraction multi-occupancy)」=
P=特定の細胞が、試みてもウェルに適合しないであろう確率(干渉の尺度)
N=ウェルの数
L=標識の数=バーコード
C=細胞の数)
本明細書に開示されている通り、本装置は、PCRエラー等の増幅エラーを制御する目的で用いることができる。例えば、核酸試料は、装置のマイクロウェルへと区分化することができる。区分化後に、試料は、マイクロウェル内でPCR増幅反応に付すことができる。マイクロウェル内のPCR産物は、本明細書に記載されている方法を用いて、同じ固有の識別子でタグ付けすることができる。産物が後に配列決定され、配列の差を実証する場合、同じ識別子を有する産物間の差は、PCRエラーに起因し得る。
他の適用において、装置を用いて、多くの場合は細胞毎の試料における遺伝子産物(例えば、タンパク質、mRNA)発現レベルを検出することができる。試料は、個々の細胞、細胞から得たmRNA抽出物のプールまたは遺伝子産物の他の収集物を含むことができる。一部の事例において、単一細胞をマイクロウェルに充填することができる。他の事例において、所望の含量のmRNA分子が個々のマイクロウェルに充填されるように、mRNAまたは他の遺伝子産物のプールを充填することができる。
本明細書に用いられている用語法は、特定の実施形態の説明のみを目的としており、本開示の装置の限定を目的としない。本明細書において、文脈が明らかにそれ以外を示すのでなければ、単数形(「a」、「an」および「the」)は、複数形を同様に含むことを目的とする。さらに、用語「含む(including)」、「含む(includes)」、「有する(having)」、「有する(has)」、「有する(with)」またはこれらの異形が、詳細な説明および/または特許請求の範囲において用いられる程度まで、斯かる用語は、用語「含む(comprising)」と同様の様式で包括的であることを目的とする。
マイクロウェルカプセルアレイを調製して、血液試料から得た個々のヒトB細胞において核酸配列決定を行う。およそ15,000個の細胞を収集し、装置への充填に用いた。150,000個のマイクロウェルを含有する本開示の装置を用いる。各ウェルは、125umの直径および125umの高さを有する円柱状の形状をしており、ウェル当たり最大で1個のカプセルの充填を可能にする。装置に充填するためのマイクロカプセルが100umの直径を有するように、PNIPAMハイドロゲル殻壁を有するエマルション重合で作製したマイクロカプセルを作製する。PNIPAM殻が磁鉄粒子を含有するように、マイクロカプセルを作製する。次に、殻の外表面を、B細胞外部における膜貫通B細胞受容体に特異的な抗体に化学的にカップリングする。
マイクロウェルカプセルアレイを調製して、ヒト皮膚細胞の集団から単離した個々のDNA鎖において核酸配列決定を行う。洗剤および熱を用いて細胞を溶解し、クロロホルム/エタノール抽出によりおよそ15,000コピーの二倍体DNAを沈殿させる。およそ10,000コピーの一倍体DNAを有するDNAの再懸濁物を装置に充填する。300,000個のマイクロウェルを有する本開示の装置を用いる。各ウェルは、125umの直径および125umの高さを有する円柱状の形状であり、ウェル当たり最大で1個のカプセルを充填させることができる。装置へと充填するために、PNIPAMハイドロゲル殻壁を有するエマルション重合により作製されたマイクロカプセルを、100umの直径を有する球の規格となるよう作製する。
110 殻層
120 内部区画(内部層)
130 エンベロープ層(第2の層)
140 より小型のマイクロカプセル
150 ポリマー材料
160 非混和性流体
170 非混和性流体の層
180 内部マイクロカプセル
200 入口
210 出口
220 プレート
240 入口
250 流路
260 出口
270 マイクロウェル
410 試薬
420 核酸分析物
440 インキュベーション工程
450 試薬
460 マイクロカプセル
Claims (51)
- 液滴を含む組成物であって、
a. 前記液滴が、ビーズと、化学的試薬または生物学的試薬とを含み、
b. 前記ビーズが、前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に分解性となり、
c. 前記ビーズが、前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に前記ビーズから放出可能であるオリゴヌクレオチドバーコードを含む
組成物。 - 前記ビーズが、化学的クロスリンカーを含む、請求項1に記載の組成物。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項2に記載の組成物。
- 前記ビーズが、ポリマーゲルを含む、請求項1に記載の組成物。
- 前記ポリマーゲルが、ポリアクリルアミドゲルである、請求項4に記載の組成物。
- 前記ビーズが、ゲルビーズである、請求項1に記載の組成物。
- 前記化学的試薬が還元剤を含み、前記還元剤がジチオスレイトール(DTT)またはトリス(2-カルボキシエチル)ホスフィン(TCEP)である、請求項1に記載の組成物。
- 前記オリゴヌクレオチドバーコードが、ウラシルを含む、請求項1に記載の組成物。
- 前記液滴が、デオキシウリジン三リン酸(dUTP)を取り込めないポリメラーゼを含む、請求項1に記載の組成物。
- 前記液滴が、標的分析物を含む、請求項1に記載の組成物。
- 前記標的分析物が、核酸である、請求項10に記載の組成物。
- 前記核酸が、DNA、RNA、アンプリコン、合成ポリヌクレオチド、ポリヌクレオチド、オリゴヌクレオチド、cDNA、dsDNA、ssDNA、プラスミドDNA、コスミドDNA、高分子量(MW)DNA、染色体DNA、ゲノムDNA、ウイルスDNA、細菌DNA、mtDNA(ミトコンドリアDNA)、mRNA、rRNA、tRNA、nRNA、siRNA、snRNA、snoRNA、scaRNA、マイクロRNA、dsRNA、リボザイム、リボスイッチおよびウイルスRNAからなる群から選択される、請求項11に記載の組成物。
- 前記核酸が、ゲノムDNA(gDNA)である、請求項11に記載の組成物。
- 前記ビーズが、そこに放出可能に結合した少なくとも1,000,000個のオリゴヌクレオチドバーコードを含む、請求項1に記載の組成物。
- 前記オリゴヌクレオチドバーコードが、化学的クロスリンカーを介して前記ビーズに放出可能に結合している、請求項1に記載の組成物。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項15に記載の組成物。
- 複数の区分を含む装置であって、
a. 複数の区分の少なくとも1個の区分が、(i)オリゴヌクレオチドバーコードを含むビーズ、及び(ii) 化学的試薬または生物学的試薬を含み、
b. 前記ビーズが、前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に分解性となり、
c. 前記オリゴヌクレオチドバーコードが、前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に前記ビーズから放出可能である
装置。 - 前記区分がウェルである、請求項17に記載の装置。
- 前記区分が液滴である、請求項17に記載の装置。
- 前記ビーズが、化学的クロスリンカーを含む、請求項17に記載の装置。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項20に記載の装置。
- 前記ビーズが、ポリマーゲルを含む、請求項17に記載の装置。
- 前記ポリマーゲルが、ポリアクリルアミドゲルである、請求項22に記載の装置。
- 前記ビーズが、ゲルビーズである、請求項17に記載の装置。
- 前記化学的試薬が還元剤を含み、前記還元剤がジチオスレイトール(DTT)またはトリス(2-カルボキシエチル)ホスフィン(TCEP)である、請求項17に記載の装置。
- 前記オリゴヌクレオチドバーコードが、ウラシルを含む、請求項17に記載の装置。
- 前記区分が、デオキシウリジン三リン酸(dUTP)を取り込めないポリメラーゼを含む、請求項17に記載の装置。
- 前記区分が、標的分析物を含む、請求項17に記載の装置。
- 前記標的分析物が、核酸である、請求項28に記載の装置。
- 前記核酸が、DNA、RNA、アンプリコン、合成ポリヌクレオチド、ポリヌクレオチド、オリゴヌクレオチド、cDNA、dsDNA、ssDNA、プラスミドDNA、コスミドDNA、高分子量(MW)DNA、染色体DNA、ゲノムDNA、ウイルスDNA、細菌DNA、mtDNA(ミトコンドリアDNA)、mRNA、rRNA、tRNA、nRNA、siRNA、snRNA、snoRNA、scaRNA、マイクロRNA、dsRNA、リボザイム、リボスイッチおよびウイルスRNAからなる群から選択される、請求項29に記載の装置。
- 前記核酸が、ゲノムDNA(gDNA)である、請求項29に記載の装置。
- 前記オリゴヌクレオチドバーコードが、化学的クロスリンカーを介して前記ビーズに放出可能に結合している、請求項17に記載の装置。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項32に記載の装置。
- 試料調製のための方法であって、
a. (i)オリゴヌクレオチドバーコードを含むビーズと、(ii)標的分析物と、(iii)化学的試薬または生物学的試薬とを区分へと組み合わせる工程であって、前記ビーズが、前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に分解性となる工程と、
b. 前記ビーズに対する前記化学的試薬または生物学的試薬の適用の際に、前記ビーズから前記区分へと前記オリゴヌクレオチドバーコードを放出させる工程と
を含む方法。 - 前記区分が、ウェルである、請求項34に記載の方法。
- 前記区分が、液滴である、請求項34に記載の方法。
- 前記ビーズが、ポリマーゲルを含む、請求項34に記載の方法。
- 前記ポリマーゲルが、ポリアクリルアミドである、請求項37に記載の方法。
- 前記ビーズが、ゲルビーズである、請求項34に記載の方法。
- 前記ビーズが、化学的クロスリンカーを含む、請求項34に記載の方法。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項40に記載の方法。
- 前記化学的試薬が還元剤を含み、前記還元剤がジチオスレイトール(DTT)またはトリス(2-カルボキシエチル)ホスフィン(TCEP)である、請求項34に記載の方法。
- 前記オリゴヌクレオチドバーコードが、ウラシルを含む、請求項34に記載の方法。
- 前記区分が、デオキシウリジン三リン酸(dUTP)を取り込めないポリメラーゼを含む、請求項34に記載の方法。
- 前記オリゴヌクレオチドバーコードを前記標的分析物に付着させる工程をさらに含む、請求項34に記載の方法。
- 前記付着工程が、核酸増幅反応により完了する、請求項45に記載の方法。
- 前記標的分析物が、核酸である、請求項34に記載の方法。
- 前記核酸が、DNA、RNA、アンプリコン、合成ポリヌクレオチド、ポリヌクレオチド、オリゴヌクレオチド、cDNA、dsDNA、ssDNA、プラスミドDNA、コスミドDNA、高分子量(MW)DNA、染色体DNA、ゲノムDNA、ウイルスDNA、細菌DNA、mtDNA(ミトコンドリアDNA)、mRNA、rRNA、tRNA、nRNA、siRNA、snRNA、snoRNA、scaRNA、マイクロRNA、dsRNA、リボザイム、リボスイッチおよびウイルスRNAからなる群から選択される、請求項47に記載の方法。
- 前記核酸が、ゲノムDNA(gDNA)である、請求項47に記載の方法。
- 前記オリゴヌクレオチドバーコードが、化学的クロスリンカーを介して前記ビーズに放出可能に結合している、請求項34に記載の方法。
- 前記化学的クロスリンカーが、ジスルフィド結合である、請求項50に記載の方法。
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