JP2018510662A - 生物学的試料の空間識別されるマルチプレックスな核酸分析 - Google Patents
生物学的試料の空間識別されるマルチプレックスな核酸分析 Download PDFInfo
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Abstract
Description
現在、がんはゲノムの疾患と言われている。多くの腫瘍学者およびがん研究者は、ゲノム分析ツールの進歩が早期発見および処置への道を提供することを期待している。しかしながら、これらのツールは、ほとんどの腫瘍学者が容易に入手できるレベルにまだ達していない研究所においてより重要である。改善が必要である。
診断時、全てのがん患者はモザイクであると言われている。がん患者は、少なくとも2つの別個のゲノム、すなわちがん患者が持って生まれたゲノムと、がん患者が不本意ながらがんにより獲得したゲノムとを有するため、モザイクである。さらに、腫瘍が成長するにつれて、別個のがん細胞集団が顕在化する。それにより、腫瘍内でモザイクがより一層複雑になる。このがん細胞の不均質性はしばしば、がん療法に異なって応答する細胞の部分集団を生じる。最終結果は、多くの場合、細胞のある部分集団の初期の陽性応答であり、そのため患者の腫瘍の縮小が観察されるが、その後、結局は腫瘍組織の再成長、一部のケースでは転移に至る。腫瘍の早期発見にもかかわらず、処置に抵抗性を有する細胞の部分集団を同定できなければ、高悪性度のがんを処置するのに要する時間の損失を引き起こす可能性がある。これは、患者にとって感情的にも物理的にも有害事象を引き起こす。
本明細書で使用される用語は、別段の規定がない限り関連分野におけるその通常の意味で解釈されるものとする。本明細書で使用される数種の用語およびその意味は、以下に記載の通りである。
本明細書で使用される場合、用語「伸長する」は、核酸に関して使用される場合、核酸への少なくとも1つのヌクレオチドまたはオリゴヌクレオチドの付加を意味することが意図される。特定の実施形態において、1つまたは複数のヌクレオチドを、例えばポリメラーゼ触媒作用(例えばDNAポリメラーゼ、RNAポリメラーゼまたは逆転写酵素)を介して核酸の3’末端に付加することができる。1つまたは複数のヌクレオチドを核酸の3’または5’末端に付加するのに、化学的または酵素的方法を使用することができる。1つまたは複数のオリゴヌクレオチドを、例えば化学的または酵素的な(例えばリガーゼ触媒作用)方法を介して核酸の3’または5’末端に付加することができる。核酸は、テンプレート指向的な方式で伸長させることができ、それによって伸長産物は、伸長される核酸にハイブリダイズされるテンプレートの核酸に相補的である。
上記の定義を考慮して、以下に記載された実施形態および特許請求の範囲で列挙された実施形態を理解することができる。
高密度のアレイは、約15μm未満の平均ピッチを有することを特徴とする。中密度のアレイは、約15〜30μmの平均ピッチを有し、一方で低密度のアレイは、30μmより大きい平均ピッチを有する。本発明において有用なアレイは、100μm、50μm、10μm、5μm、1μmまたは0.5μm未満の平均ピッチを有していてもよい。本明細書の上記または他所に記載の平均ピッチの値および範囲は、規則正しいアレイまたはランダムなアレイに適用可能であることが意図される。
ビーズに付着される核酸プローブは、バーコード配列を包含していてもよい。ビーズの集団は、各ビーズが1つのみのタイプのバーコードに付着されて、集団中にそれぞれ異なるバーコードを有する多くの様々なビーズが存在するように設計されていてもよい。この実施形態において、固体支持体上にビーズをランダムに分配させることは、固体支持体上に核酸プローブ(およびそれらそれぞれのバーコード配列)をランダムに配置することになると予想される。一部のケースにおいて、集団中に冗長性が存在するように、同じバーコード配列を有する複数のビーズが存在していてもよい。ビーズ集団中の固有のバーコードの数より多くのキャパシティーを有する固体支持体上にビーズの冗長な集団をランダムに分配させることは、固体支持体上でバーコードの冗長性をもたらすことになると予想される。代替として、固体支持体上におけるバーコードの集団に関して冗長ではないアレイを生産するために、ビーズの集団中の異なるバーコードの数は、固体支持体のキャパシティーを超えていてもよい。固体支持体のキャパシティーは、一部の実施形態において、ビーズを付着させるかまたはそれ以外の方法でビーズを受け入れるフィーチャ(例えば単一のビーズを占有するウェル)の数によって決定されると予想される。
本明細書に記載の方法は、様々な増幅技術のいずれも使用することができる。使用できる例示的な技術としては、これらに限定されないが、ポリメラーゼ連鎖反応(PCR)、ローリングサークル増幅(RCA)、多置換増幅(MDA)、またはランダムプライム増幅(RPA)が挙げられる。一部の実施形態において、例えば、アレイのフィーチャが、所望のキャパシティーを有する体積でアンプリコンを含有することが可能な場合、増幅は溶液中で行うことができる。好ましくは、本発明の開示の方法で使用される増幅技術は、固相上で行われると予想される。例えば、1つまたは複数のプライマー種(例えば、核酸プローブ中に存在する1つまたは複数のユニバーサルプライマー結合性部位のためのユニバーサルプライマー)が、固体支持体に付着されていてもよい。PCRの実施形態において、増幅に使用されるプライマーの一方または両方が、固体支持体に付着されていてもよい(例えばゲルを介して)。固体支持体に付着した2種のプライマーを利用する様式はしばしば、二本鎖アンプリコンが、コピーされたテンプレート配列を端部に有する2つの表面に付着したプライマー間で架橋様の構造を形成するため、架橋増幅と称される。架橋増幅に使用することができる例示的な試薬および条件は、例えば、それぞれが参照により本明細書に組み入れられる米国特許第5,641,658号、7,115,400号、もしくは8,895,249号;または米国特許公開第2002/0055100号、2004/0096853号、2004/0002090号、2007/0128624号または2008/0009420号に記載されている。固相PCR増幅はまた、固体支持体に付着した増幅プライマーの1つおよび溶液中の第2のプライマーを用いても行うことができる。表面に付着したプライマーと可溶性プライマーとの組合せを使用する例示的な様式は、エマルジョンPCRに使用される様式であり、これらは、例えば、それぞれが参照により本明細書に組み入れられるDressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003)、WO05/010145、または米国特許出願公開第2005/0130173号または2005/0064460号に記載されている。エマルジョンPCRは、この様式の例示であり、本明細書に記載の方法の目的で、エマルジョンの使用は任意選択であり、数々の実施形態にとって実際には、エマルジョンは使用されないことが理解されると予想される。
一部のシーケンシングの実施形態は、伸長産物にヌクレオチドが取り込まれたときに放出されたプロトンの検出を包含する。例えば、放出されたプロトンの検出に基づくシーケンシングは、Ion Torrent(Guilford、CT、Life TechnologiesおよびThermo Fisherの子会社)から市販されている電気的検出器および関連する技術、またはそれぞれが参照により本明細書に組み入れられる米国特許出願公開第2009/0026082号;2009/0127589号;2010/0137143号;もしくはUS2010/0282617号に記載されているシーケンシング方法およびシステムを使用することができる。
生物学的試料は、上記の細胞、細胞内要素、ウイルスまたはウイロイドの均一な培養物または集団であり得ることが理解されると予想される。代替として、生物学的試料は、細胞、細胞内要素、ウイルスまたはウイロイドの同質ではない収集物、例えば共同体または生態系の数種の異なる生物由来の収集物であり得る。例示的な共同体は、哺乳動物などの多細胞生物の消化器系、肺または他の器官中に存在する細菌の収集物である。
本明細書における使用のための組織サンプルは、組織構造の完全性を維持または保存するのに好適な温度で、例えば−20℃未満で急速冷凍することによって固定することができる。別の例において、組織は、当業界において公知のホルマリン固定およびパラフィン包埋(FFPE)方法を使用して調製することができる。要求に応じて他の固定剤および/または包埋材料を使用することができる。固定または包埋した組織サンプルは、公知の方法を使用して、切片化する、すなわち薄くスライスすることができる。例えば、組織サンプルは、組織サンプルの構造的な完全性とサンプル中の核酸の化学特性の両方を維持するのに好適な温度に設定した冷却したマイクロトームまたはクライオスタットを使用して切片化することができる。
生物学的試料に関して特に関連性のある源は、ヒトである。試料は、器官に由来するものであってもよく、例えば、筋骨格系の器官、例えば筋肉、骨、腱または靱帯;消化器系の器官、例えば唾液腺、咽頭、食道、胃、小腸、大腸、肝臓、胆嚢または膵臓;呼吸器系の器官、例えば喉頭、気管、気管支、肺または横隔膜;泌尿器系の器官、例えば腎臓、尿管、膀胱または尿道;生殖器、例えば卵巣、ファロピウス管、子宮、膣、胎盤、睾丸、精巣上体、精管、精嚢、前立腺、陰茎または陰嚢;内分泌系の器官、例えば下垂体、松果腺、甲状腺、副甲状腺、または副腎;循環系の器官、例えば心臓、動脈、静脈または毛細血管;リンパ系の器官、例えばリンパ管、リンパ節、骨髄、胸腺または脾臓;中枢神経系の器官、例えば脳、脳幹、小脳、脊髄、脳神経、または脊髄神経;感覚器官、例えば目、耳、鼻、または舌;または外皮の器官、例えば皮膚、皮下組織または乳腺などに由来するものであってもよい。一部の実施形態において、生物学的試料は、体液または排泄物、例えば血液、リンパ液、涙、汗、唾液、精液、膣分泌液、耳垢、排泄物または尿から得られる。
マルチプレックスの実施形態において、複数の異なる核酸プローブは、生物学的試料からの異なる標的核酸配列にハイブリダイズする異なる標的捕獲配列を包含していてもよい。異なる標的捕獲配列は、生物学的試料からの1つまたは複数の所望の標的核酸に選択的に結合させるのに使用することができる。一部のケースにおいて、異なる核酸プローブは、固体支持体上のプローブの全てまたはサブセットに共通の標的捕獲配列を包含していてもよい。例えば、固体支持体上の核酸プローブは、ポリAまたはポリT配列を有していてもよい。このようなプローブまたはそのアンプリコンは、ポリAまたはポリTテールを有するmRNA分子、cDNA分子またはそのアンプリコンにハイブリダイズすることができる。mRNAまたはcDNA種は異なる標的配列を有すると予想されるが、捕獲は、共通のポリAまたはポリT配列領域によって媒介されると予想される。
有用な他の標的捕獲成分としては、例えば、固体支持体に核酸プローブを付着させるのに有用なものとして本明細書に記載された成分が挙げられる。
本明細書に記載の方法は、固体支持体と接触する生物学的試料の画像を獲得する工程をさらに包含していてもよい。固体支持体は、本明細書に記載の様々な状況のいずれにおけるものでもよい。例えば、固体支持体は、付着された核酸プローブまたは付着された核酸プローブ由来のクラスターを包含していてもよい。代替として、固体支持体は、核酸プローブを包含していなくてもよく、その代わりに核酸プローブが付着する前の状況または固体支持体から核酸プローブを除去した後の状況であってもよい。したがって、本明細書に記載の方法における様々なポイントのいずれかで、画像を得ることができる。
核酸を切断するのに使用できる切断部位および方法の他の例は、例えば参照により本明細書に組み入れられる米国特許第7,960,120号に記載されている。
固体支持体から放出される改変された核酸プローブ(例えば伸長した核酸プローブ)をプールして、流体混合物を形成することができる。混合物は、例えば、少なくとも10、100、1×103、1×104、1×105、1×106、1×107、1×108、1×109個またはそれより多くの異なる改変されたプローブを包含していてもよい。代替として、またはそれに加えて、流体混合物は、最大で1×109、1×108、1×107、1×106、1×105、1×104、1×103、100、10個またはそれより少ない異なる改変されたプローブを包含していてもよい。流体混合物は、改変された核酸プローブの検出が可能なように操作されていてもよい。例えば、改変された核酸プローブは、第2の固体支持体(すなわち生物学的試料と接触させて改変された後に核酸プローブが放出された固体支持体とは異なる)上で空間的に分離されていてもよく、またはプローブは、流体ストリーム中で一時的に分離されていてもよい。
Illuminaフローセルを使用して組織サンプルからのmRNAの空間的タグ付け
図1に、バーコード化されたオリゴ−dTを含有するクラスターを生成し、次いでバーコード化されたオリゴ−dTを制限酵素消化によって露出させ、続いてシーケンシングするための方法を説明する。一本鎖のバーコード化されたオリゴ−dA、P5’、P7、SBS3シーケンシングプライマー結合性部位およびBspHI制限酵素部位(図1の上のパネルに示される)を含有するフラグメントのライブラリーを、オリゴ合成(Integrated DNA Technologies)によって調製した。バーコードは27merであり、合成中にランダムに生成された。シーケンシングによるバーコードのデコードのために、SBS3シーケンシングプライマーのための結合性部位を包含させた。クラスター形成および線状化のときにオリゴdT部位が生成するように、オリゴ−dAストレッチを包含させた。Illumina GAフローセルでの標準的なクラスター化学(Illumina TruSeq PE Cluster Kit v3 cBot P/N:15037931)に従って、製造元の推奨されたプロトコールを使用して、架橋増幅およびクラスター形成を実行した。
Illuminaフローセル上への細胞接着
単一の細胞を、パターン化したフローセル上で捕獲した(HiSeq X10フローセル、Illumina)。全ての試薬のフロー工程を、蠕動ポンプまたはcBOTクラスター生成機器(Illumina)を使用して実行した。簡単に述べると、パターン化したフローセルの全てのレーン上に、ヌクレアーゼ非含有水、続いて30〜70KのポリDリシン溶液(100μg/mlおよび20μg/ml)を100μl/分の流速で8分流した。加熱不活性化したウシ胎児血清(Life Technologies、番号10082−139)も接着剤として試験した。接着剤をフローセルのレーン上で1時間インキュベートし、続いて1×PBS+0.5%Pluronic F−68(Life Technologies、番号24040−032)洗浄液をインキュベートした。次に、100μl/分の速度で細胞5〜50個/μlまたはおよそ細胞100〜1000個/レーンで流動させ、続いて細胞を結合させるために60分インキュベーション工程を行うことによって、コーティングしたフローセルに細胞を接着させた。75μl/分の速度で1×PBS/0.5%Pluronicを用いてフローセルを洗浄した。フローセル上に細胞が固定された場合、上述したように細胞を流動させた後にフローセル上に1%パラホルムアルデヒド(PFA)を流し、15分インキュベートし、続いて1×PBS/0.5%Pluronicの工程を行った。フローセルを除去し、顕微鏡を使用してレーン当たりの細胞の数を数えた。
ゲルに付着したプローブ表面による標的mRNAの空間的に局所的な捕獲
この例は、ゲルコーティングしたスライド上のポリTプローブのローンの作出、ポリTプローブのローン上部への組織切片の設置、組織切片からのRNAの放出、ポリTプローブによる放出されたmRNAの捕獲、ポリTプローブをCy3標識するための逆転写、組織の除去、およびスライドの画像化を説明する。
図7のパネルAは、ゲルに付着したプローブを作り出すのに使用される工程および試薬の図解的な表示を示す。簡単に述べると、顕微鏡のスライドをシラン非含有アクリルアミド(SFA)でコーティングし、P5およびP7プライマーを付着させ(参照により本明細書に組み入れられる米国特許出願公開第2011/0059865号を参照)、ポリA配列およびP5またはP7相補配列のいずれかを有するプローブを、それぞれP5およびP7プライマーにハイブリダイズさせ、P5およびP7プライマーを伸長させて、ポリT配列伸長を生産した。Cy5で標識されたポリAオリゴヌクレオチドを伸長したプライマーにハイブリダイズさせて、Axonイメージャーを使用して表面を画像化することによって、品質管理工程を実行した。
図7の画像で示されるように、mRNA種を放出した組織のエリアの近位のゲルのエリアは蛍光で表され、mRNAを放出しなかったエリアは画像中で暗部として表される。したがって、捕獲されたmRNAは、組織のフィンガープリント様の画像を作り出した。
BeadArray(商標)表面に付着したプローブによる標的mRNAの空間的に局所的な捕獲
この例は、ポリTプローブを有するBeadArray(商標)の上部への組織切片の設置、組織切片からのRNAの放出、ポリTプローブによる放出されたmRNAの捕獲、ポリTプローブをCy5標識するための逆転写、組織の除去、およびBeadArray(商標)の画像化を説明する。
図8のパネルAで示されるように、マウス嗅覚組織切片を、ポリTプローブを有するBeadArray(商標)に設置した。組織を処理して、mRNAを放出させ、放出されたmRNAのポリAテールを、プローブのポリT配列にハイブリダイズさせた。テンプレートとして捕獲されたmRNAを使用してポリT配列を伸長させ、伸長したプライマーをCy5で選択的に標識した。BeadArray(商標)から組織を除去し、BeadArray(商標)を画像化して、Cy5蛍光(flourescence)を検出した。
本出願にわたり、様々な刊行物、特許または特許出願が参照されている。本出願において、これらの刊行物の開示は、参照によりその全体が本明細書に組み入れられる。
数々の実施形態が記載されている。それにもかかわらず、様々な改変がなされ得ることが理解されると予想される。したがって、他の実施形態は、以下の特許請求の範囲内である。
Claims (105)
- 生物学的試料の核酸を空間的にタグ付けするための方法であって、
(a)固体支持体に異なる核酸プローブを付着させて、固体支持体上でランダムに配置されたプローブを生産する工程であって、異なる核酸プローブはそれぞれ、バーコード配列を含み、ランダムに配置されたプローブのそれぞれは、固体支持体上に他のランダムに配置されたプローブとは異なるバーコード配列を含む、工程;
(b)固体支持体上で核酸検出反応を実行して、固体支持体上でランダムに配置されたプローブのバーコード配列を決定する工程;
(c)生物学的試料を、ランダムに配置されたプローブを含む固体支持体と接触させる工程;
(d)ランダムに配置されたプローブを、ランダムに配置されたプローブの近位にある生物学的試料の部分からの標的核酸にハイブリダイズさせる工程;および
(e)ランダムに配置されたプローブを伸長させて、バーコード配列と標的核酸からの配列とを含む伸長したプローブを生産し、それによって生物学的試料の核酸を空間的にタグ付けする工程
を含む方法。 - 工程(a)が、固体支持体上でランダムに配置されたプローブのそれぞれを増幅して、固体支持体上に核酸プローブの複数のアンプリコンを含むクラスターを形成することをさらに含む、請求項1に記載の方法。
- 固体支持体が、複数のクラスターを含み、複数のクラスターの平均ピッチが、10μm未満である、請求項2に記載の方法。
- 固体支持体が、複数のクラスターを含み、クラスターの平均面積が、100μm2未満である、請求項2または3に記載の方法。
- 核酸検出反応が、シーケンシング反応を含む、請求項1から4までのいずれか1項に記載の方法。
- 核酸検出反応が、デコーダープローブのハイブリダイゼーション反応を含む、請求項1に記載の方法。
- 固体支持体が、ビーズのアレイを含み、異なる核酸プローブが、アレイ中の異なるビーズに付着される、請求項1から6までのいずれか1項に記載の方法。
- 工程(a)が、固体支持体上に異なるビーズをランダムに分配させることを含む、請求項7に記載の方法。
- 固体支持体が、複数の前記ビーズの1個のビーズのみを受け入れる寸法を有するウェルを含む、請求項7に記載の方法。
- 異なるビーズが、異なるバーコード配列に付着されており、異なるバーコード配列の数が、ウェルの数を超える、請求項9に記載の方法。
- 固体支持体が、少なくとも1×106個の、異なる核酸プローブを含む異なるビーズを含む、請求項7から10までのいずれか1項に記載の方法。
- ビーズのアレイが、10μm未満の平均ピッチを含む、請求項7から11までのいずれか1項に記載の方法。
- ビーズが、10μm未満の平均直径を含む、請求項7から12までのいずれか1項に記載の方法。
- 固体支持体に異なる核酸プローブを付着させることが、異なる核酸プローブの流体混合物を固体支持体と接触させることを含む、請求項1から13までのいずれか1項に記載の方法。
- 固体支持体が、別個のフィーチャのパターンを含み、異なる核酸プローブが、パターン中の別個のフィーチャにランダムに分配される、請求項14に記載の方法。
- 異なるバーコード配列の数が、流体混合物と接触するフィーチャの数を超える、請求項15に記載の方法。
- 固体支持体が、少なくとも1×106個の異なる核酸プローブを含む、請求項1または請求項14から16のいずれか1項に記載の方法。
- 固体支持体が、ゲルコーティングを含む、請求項1から17までのいずれか1項に記載の方法。
- 複数の核酸プライマーが、ゲルコーティングに付着されており、複数のもののうちの核酸プライマーが、複数のもののうちの核酸プライマーに共通のユニバーサルプライマー配列を含む、請求項18に記載の方法。
- 異なる核酸プローブが、ユニバーサルプライマー配列にハイブリダイズするユニバーサルプライマー結合性配列を含む、請求項19に記載の方法。
- 工程(a)が、核酸プライマーの伸長によって異なる核酸プローブを増幅し、それによって固体支持体上に核酸プローブの複数のアンプリコンを含む核酸クラスターを生産することをさらに含む、請求項20に記載の方法。
- 第2の複数の核酸プライマーが、ゲルコーティングに付着されており、第2の複数のもののうちの核酸プライマーが、第2の複数のもののうちの核酸プライマーに共通の第2のユニバーサルプライマー配列を含む、請求項21に記載の方法。
- 異なる核酸プローブが、第2のユニバーサルプライマー配列にハイブリダイズする第2のユニバーサルプライマー結合性配列を含み、増幅することは、架橋増幅を含む、請求項22に記載の方法。
- 異なる核酸プローブが、生物学的試料からの異なる標的核酸配列にハイブリダイズする異なる標的捕獲配列を含む、請求項1から23までのいずれか1項に記載の方法。
- 異なる核酸プローブが、固体支持体上でランダムに配置されたプローブに共通の標的捕獲配列を含む、請求項1から23までのいずれか1項に記載の方法。
- 標的捕獲配列が、ポリTまたはポリA配列を含む、請求項25に記載の方法。
- 固体支持体と接触する生物学的試料の画像を獲得する工程をさらに含む、請求項1から26までのいずれか1項に記載の方法。
- ランダムに配置されたプローブで決定されたバーコード配列を、生物学的試料の画像における配置と相関させる工程をさらに含む、請求項27に記載の方法。
- 固体支持体から伸長したプローブを除去する工程をさらに含む、請求項1、27または28に記載の方法。
- 伸長したプローブをプールして、固体支持体から除去された伸長したプローブの混合物を形成する工程をさらに含む、請求項29に記載の方法。
- 固体支持体から除去された伸長したプローブの標的核酸からの配列およびバーコード配列を決定する工程をさらに含む、請求項29または30に記載の方法。
- 固体支持体から除去された伸長したプローブを、第2の固体支持体に付着させる工程をさらに含む、請求項29、30または31に記載の方法。
- 標的核酸からの配列およびバーコード配列が、合成によるシーケンシング技術によって決定される、請求項31または32に記載の方法。
- 工程(b)の間、固体支持体が、フローセル中またはその上に配置されている、請求項1から33までのいずれか1項に記載の方法。
- 工程(c)の間、固体支持体が、フローセルから除去される、請求項34に記載の方法。
- 工程(c)の間、フローセルを開放して、固体支持体を露出させる、請求項34に記載の方法。
- 固体支持体と接触させる生物学的試料が、細胞の混合物である、請求項1から36までのいずれか1項に記載の方法。
- 工程(c)が、細胞を固体支持体に付着させることをさらに含む、請求項37に記載の方法。
- 工程(c)が、細胞を溶解させて、細胞から標的核酸を放出させることをさらに含む、請求項37または38に記載の方法。
- 固体支持体と接触させる生物学的試料が、組織である、請求項1から36までのいずれか1項に記載の方法。
- 工程(c)が、組織を固体支持体に付着させることをさらに含む、請求項40に記載の方法。
- 工程(c)が、組織を透過化して、組織から標的核酸を放出させることをさらに含む、請求項40または41に記載の方法。
- 標的核酸が、mRNA、gDNA、rRNAまたはtRNAからなる群より選択される、請求項1から42までのいずれか1項に記載の方法。
- 生物学的試料の核酸を空間的にタグ付けするための方法であって、
(a)固体支持体に付着した複数の核酸プライマーを提供する工程であって、複数のもののうちの核酸プライマーは、複数のもののうちの核酸プライマーに共通のユニバーサルプライマー配列を含む、工程;
(b)核酸プローブの集団を、複数の核酸プライマーに結合させる工程であって、核酸プローブは、ユニバーサルプライマー配列にハイブリダイズするユニバーサルプライマー結合性配列、標的捕獲配列および集団中の他の核酸プローブのバーコード配列とは異なるバーコード配列を含み、それによって固体支持体上でランダムに配置された位置に異なる核酸プローブを付着させる、工程;
(c)核酸プライマーの伸長によって異なる核酸プローブを増幅し、それによって固体支持体上でランダムに配置された位置で、バーコード配列のコピーと標的捕獲配列とを有する核酸クラスターを生産する工程;
(d)シーケンシング反応を実行して、固体支持体上でランダムに配置された位置におけるバーコード配列を決定する工程;
(e)生物学的試料を、固体支持体上で核酸クラスターと接触させる工程;
(f)クラスターの標的捕獲配列を、クラスターの近位にある生物学的試料の部分からの標的核酸にハイブリダイズさせる工程;および
(g)標的捕獲配列を伸長させて、標的核酸からの配列とバーコード配列のコピーとを含む伸長したプローブを生産し、それによって生物学的試料の核酸をタグ付けする工程
を含む、方法。 - 第2の複数の核酸プライマーが、固体支持体に付着されており、第2の複数のもののうちの核酸プライマーが、第2の複数のもののうちの核酸プライマーに共通の第2のユニバーサルプライマー配列を含む、請求項44に記載の方法。
- 異なる核酸プローブが、第2のユニバーサルプライマー配列にハイブリダイズする第2のユニバーサルプライマー結合性配列を含む、請求項45に記載の方法。
- 異なる核酸プローブを増幅することが、架橋増幅を含み、第1および第2の複数のもののうちの核酸プライマーが、伸長される、請求項46に記載の方法。
- 核酸プローブが、標的捕獲配列と第2のユニバーサルプライマー結合性配列との間に切断部位をさらに含む、請求項45から47までのいずれか1項に記載の方法。
- 工程(g)の前に、標的捕獲配列と第2のユニバーサルプライマー結合性配列との間の切断部位を切断する工程をさらに含む、請求項48に記載の方法。
- 異なる核酸プローブが、生物学的試料からの異なる標的核酸配列にハイブリダイズする異なる標的捕獲配列を含む、請求項44から49までのいずれか1項に記載の方法。
- 異なる核酸プローブが、固体支持体上でランダムに配置されたプローブに共通の標的相補配列を含む、請求項44から49までのいずれか1項に記載の方法。
- 標的相補配列が、ポリTまたはポリA配列を含む、請求項51に記載の方法。
- 標的核酸が、工程(f)において標的相補配列にハイブリダイズするポリAテールを有するmRNA分子を含む、請求項51に記載の方法。
- 核酸プローブのそれぞれが、バーコード配列と核酸プローブの固体支持体への付着点との間に配置される切断部位を含む、請求項44から53までのいずれか1項に記載の方法。
- バーコード配列と核酸プローブの固体支持体への付着点との間に配置される切断部位で切断することによって、固体支持体から伸長したプローブを除去する工程をさらに含む、請求項54に記載の方法。
- 伸長したプローブをプールして、固体支持体から除去された伸長したプローブの混合物を形成する工程をさらに含む、請求項55に記載の方法。
- 固体支持体から除去された伸長したプローブの標的核酸からの配列およびバーコード配列を決定する工程をさらに含む、請求項55または56に記載の方法。
- 固体支持体から除去された伸長したプローブを、第2の固体支持体に付着させる工程をさらに含む、請求項55、56または57に記載の方法。
- 標的核酸からの配列およびバーコード配列が、合成によるシーケンシング技術によって決定される、請求項57または58に記載の方法。
- 異なる核酸プローブが、シーケンシングプライマー結合性部位をさらに含み、合成によるシーケンシング技術が、シーケンシングプライマーをシーケンシングプライマー結合性部位にハイブリダイズすることを含む、請求項59に記載の方法。
- シーケンシングプライマー結合性部位の配列が、異なる核酸プローブで同じである、請求項60に記載の方法。
- 固体支持体が、ゲルコーティングを含む、請求項44から61までのいずれか1項に記載の方法。
- 核酸プライマーが、ゲルを介して固体支持体に付着されている、請求項62に記載の方法。
- 固体支持体が、複数のクラスターを含み、複数のクラスターの平均ピッチが、10μm未満である、請求項44から63までのいずれか1項に記載の方法。
- 固体支持体が、複数のクラスターを含み、クラスターの平均直径が、100μm2未満である、請求項44から64までのいずれか1項に記載の方法。
- 工程(b)が、異なる核酸プローブの流体混合物を固体支持体と接触させることを含む、請求項44から65までのいずれか1項に記載の方法。
- 固体支持体が、別個のフィーチャのパターンを含み、異なる核酸プローブが、パターン中の別個のフィーチャにランダムに分配される、請求項44から66までのいずれか1項に記載の方法。
- 異なるバーコード配列の数が、流体混合物と接触するフィーチャの数を超える、請求項67に記載の方法。
- 固体支持体が、少なくとも1×106個の異なる核酸プローブを含む、請求項44から68までのいずれか1項に記載の方法。
- 固体支持体と接触する生物学的試料の画像を獲得する工程をさらに含む、請求項44から66までのいずれか1項に記載の方法。
- ランダムに配置された位置で決定されたバーコード配列を、生物学的試料の画像における配置と相関させる工程をさらに含む、請求項70に記載の方法。
- 工程(d)の間、固体支持体が、フローセル中またはその上に配置されている、請求項44から71までのいずれか1項に記載の方法。
- 工程(e)の間、固体支持体が、フローセルから除去される、請求項72に記載の方法。
- 工程(e)の間、フローセルを開放して、固体支持体を露出させる、請求項72に記載の方法。
- 固体支持体と接触させる生物学的試料が、細胞の混合物である、請求項44から74までのいずれか1項に記載の方法。
- 工程(e)が、細胞を固体支持体に付着させることをさらに含む、請求項75に記載の方法。
- 工程(e)が、細胞を溶解させて、細胞から標的核酸を放出させることをさらに含む、請求項75または76に記載の方法。
- 固体支持体と接触させる生物学的試料が、組織である、請求項44から77までのいずれか1項に記載の方法。
- 工程(e)が、組織を固体支持体に付着させることをさらに含む、請求項78に記載の方法。
- 工程(e)が、組織を透過化して、組織から標的核酸を放出させることをさらに含む、請求項78または79に記載の方法。
- 標的核酸が、mRNA、gDNA、rRNAまたはtRNAからなる群より選択される、請求項44から80までのいずれか1項に記載の方法。
- 生物学的試料の核酸を空間的にタグ付けするための方法であって、
(a)固体支持体上にビーズのアレイを提供する工程であって、異なる核酸プローブは、アレイ中の異なるビーズに付着され、異なる核酸プローブはそれぞれ、バーコード配列を含み、各ビーズは、固体支持体上に他のビーズからの異なるバーコード配列を含み、異なる核酸プローブのそれぞれは、標的捕獲配列を含む、工程;
(b)固体支持体上でデコーダープローブのハイブリダイゼーション反応を実行して、固体支持体上でランダムに配置されたプローブにおけるバーコード配列を決定する工程;
(c)生物学的試料をビーズのアレイと接触させる工程;
(d)異なる核酸プローブを、ビーズの近位にある生物学的試料の部分からの標的核酸にハイブリダイズさせる工程;および
(e)異なる核酸プローブを伸長させて、標的核酸からの配列とバーコード配列とを含む伸長したプローブを生産し、それによって生物学的試料の核酸をタグ付けする工程
を含む、方法。 - 工程(a)が、固体支持体上に異なるビーズをランダムに分配させることを含む、請求項82に記載の方法。
- 固体支持体が、複数の前記ビーズの1個のビーズのみを受け入れる寸法を有するウェルを含む、請求項82または83に記載の方法。
- 異なるバーコード配列の数が、ウェルの数を超える、請求項84に記載の方法。
- ビーズのアレイが、少なくとも1×106個の、異なる核酸プローブを含む異なるビーズを含む、請求項82から85までのいずれか1項に記載の方法。
- ビーズのアレイが、10μm未満の平均ピッチを含む、請求項82から86までのいずれか1項に記載の方法
- ビーズが、10μm未満の平均直径を含む、請求項82から87までのいずれか1項に記載の方法。
- 異なる核酸プローブが、生物学的試料からの異なる標的核酸配列にハイブリダイズする異なる標的捕獲配列を含む、請求項82から88までのいずれか1項に記載の方法。
- 異なる核酸プローブが、ビーズのアレイ中の異なる核酸プローブに共通の標的捕獲配列を含む、請求項82から88までのいずれか1項に記載の方法。
- 標的捕獲配列が、ポリTまたはポリA配列を含む、請求項90に記載の方法。
- ビーズのアレイと接触する生物学的試料の画像を獲得する工程をさらに含む、請求項82から91までのいずれか1項に記載の方法。
- ビーズのバーコード配列を、生物学的試料の画像における配置と相関させる工程をさらに含む、請求項92に記載の方法。
- ビーズのアレイから伸長したプローブを除去する工程をさらに含む、請求項82、92または93に記載の方法。
- 伸長したプローブをプールして、ビーズのアレイから除去された伸長したプローブの混合物を形成する工程をさらに含む、請求項94に記載の方法。
- 固体支持体から除去された伸長したプローブの標的核酸からの配列およびバーコード配列を決定する工程をさらに含む、請求項94または95に記載の方法。
- 固体支持体から除去された伸長したプローブを、第2の固体支持体に付着させる工程をさらに含む、請求項94、95または96に記載の方法。
- 標的核酸からの配列およびバーコード配列が、合成によるシーケンシング技術によって決定される、請求項82から97までのいずれか1項に記載の方法。
- 固体支持体と接触させる生物学的試料が、細胞の混合物である、請求項82から98までのいずれか1項に記載の方法。
- 工程(c)が、細胞を固体支持体に付着させることをさらに含む、請求項99に記載の方法。
- 工程(c)が、細胞を溶解させて、細胞から標的核酸を放出させることをさらに含む、請求項99または100に記載の方法。
- 固体支持体と接触させる生物学的試料が、組織である、請求項82に記載の方法。
- 工程(c)が、組織を固体支持体に付着させることをさらに含む、請求項102に記載の方法。
- 工程(c)が、組織を透過化して、組織から標的核酸を放出させることをさらに含む、請求項102または103に記載の方法。
- 標的核酸が、mRNA、gDNA、rRNAまたはtRNAからなる群より選択される、請求項82から104までのいずれか1項に記載の方法。
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