JP2023085483A - ヌクレオチド配列決定のためのヒドロゲルビーズ - Google Patents
ヌクレオチド配列決定のためのヒドロゲルビーズ Download PDFInfo
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Abstract
Description
本願は、発明の名称「ヌクレオチド配列決定のためのヒドロゲルビーズ」の2017年8月1日に出願した米国仮特許出願第62/539,956号に対して優先権を主張し、その全体が参照により本明細書中に援用される。
1つの実施形態には、ヒドロゲルポリマーおよび遺伝物質を含むビーズが挙げられる。本明細書中で使用される「ヒドロゲル」という用語は、有機ポリマー(天然または合成)が共有結合、イオン結合、または水素結合によって架橋されて、水分子を捕捉してゲルを形成する三次元の開いた格子構造を形成するときに形成される物質を意味する。いくつかの実施形態では、ヒドロゲルは、生体適合性ヒドロゲルであってもよい。本明細書中で使用される「生体適合性ヒドロゲル」という用語は、生きた細胞に対して毒性でないゲルを形成し、取り込まれた細胞への酸素および栄養物の十分な拡散を可能にして、生存性を維持するポリマーを意味する。いくつかの実施形態では、ヒドロゲルポリマーは、水のような60~90%の流体と、10~30%のポリマーとを含む。いくらかの実施形態では、ヒドロゲルの含水率は約70~80%である。
本明細書中に提供されるいくつかの実施形態は、遺伝物質をカプセル化するビーズの製造方法に関する。いくつかの実施形態では、ヒドロゲルビーズは、渦支援エマルジョンによって調製される。本明細書中で使用される、渦支援エマルジョンとは、図1Aおよび図1Bに示されるように、管、バイアル、または反応容器などの容器内の遺伝物質とヒドロゲルポリマーの撹拌を意味する。これらの成分は、例えば手動または機械的な撹拌または振動によって混合することができる。いくつかの実施形態では、手動混合により、2、3、4、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、または150μmの直径、または上記値のいずれか2つによって規定される範囲内のサイズを有する遺伝物質をカプセル化するヒドロゲルビーズが得られる。いくつかの実施形態では、ビーズのサイズは不均一であり、従ってビーズのサイズは、様々な直径のビーズを含む。
本明細書中に提供されるいくつかの実施形態は、ビーズ内で遺伝物質を処理する方法に関する。いくつかの実施形態では、ヒドロゲルビーズ内にカプセル化された遺伝物質は、核酸処理のための1つ以上の試薬と接触される。いくつかの実施形態では、遺伝物質はヒドロゲルビーズ内に保持され、試薬はヒドロゲルビーズの細孔を通過することができる。いくつかの実施形態では、試薬は、溶解剤、核酸精製剤、DNA増幅剤、標識化剤、PCR剤、または遺伝物質の加工に使用される他の薬剤を含むことができる。このように、ヒドロゲルビーズは、ヒドロゲルビーズ内に遺伝物質自体を保持しながら、ヒドロゲルビーズの内外を試薬のバリアが通過することを可能にすることにより、ヒドロゲルビーズ内での遺伝物質の制御された反応のための微小環境を提供する。
本明細書中に提供されるシステム、方法および組成物のいくつかの実施形態は、アダプタが標的核酸に連結される方法を含む。アダプタは、配列決定プライマー結合部位、増幅プライマー結合部位、およびインデックスを含むことができる。例えば、アダプタは、P5配列、P7配列、またはそれらの相補体を含むことができる。本明細書中で使用される場合、P5配列は、配列番号1(AATGATACGGCGACCACCGA)で定義される配列を含み、P7配列は、配列番号2(CAAGCAGAAGACGGCATACGA)によって定義される配列を含む。いくつかの態様において、P5またはP7配列は、スペーサポリヌクレオチドを更に含むことができ、スペーサポリヌクレオチドは、1~20個、例えば1~15個または1~10個のヌクレオチド、例えば、長さで2、3、4、5、6、7、8、9、または10個のヌクレオチドであってもよい。いくつかの実施形態では、スペーサは10ヌクレオチドを含む。いくつかの実施形態では、スペーサは、10Tスペーサなどの、ポリTスペーサである。スペーサヌクレオチドは、ポリヌクレオチドの5’端に含まれてもよく、ポリヌクレオチドの5’端との結合によって好適な担体に結合していてもよい。結合は、ポリヌクレオチドの5’端に存在するホスホロチオエートなどの硫黄含有求核試薬によって達成することができる。いくつかの態様において、ポリヌクレオチドは、ポリTスペーサおよび5’ホスホロチオエート基を含む。従って、いくつかの実施形態では、P5配列は5’ホスホロチオエート‐TTTTTTTTTTAATGGCGACCACCGA‐3’、配列番号3であり、いくつかの実施形態では、P7配列は5’ホスホロチオエート‐TTTTTTTTTTAATGGCGACCACCGA‐3’、配列番号4である。
以下の実施例は、手動撹拌およびマイクロ流体液滴発生器を使用して、微生物細胞をカプセル化するヒドロゲルビーズを調製する実施形態を実証する。
以下の実施例は、実施例1からのヒドロゲルビーズをヌクレオチドで官能基化する方法を実証する。
以下の実施例は、架橋剤の可逆的な切断の際の、その中にカプセル化された遺伝物質を有するヒドロゲルビーズの内容物を放出する能力を実証する。
以下の実施例は、ヒドロゲルビーズ内にカプセル化された遺伝物質から核酸を配列決定する方法を実証する。
以下の実施例は、遺伝物質をカプセル化するヒドロゲルビーズからのDNAライブラリの配列決定を実証する。
以下の実施例は、ヒドロゲルビーズ内で調製されたDNAライブラリの空間インデックス化を実証する。
210 … ヒドロゲルビーズ
215 … 水性ストリーム用チャネル
220 … 不混和性流体用チャネル
Claims (39)
- ヒドロゲルポリマー;および
ヒドロゲル内に配置された遺伝物質
を含み、
ビーズが、該遺伝物質を保持しながら、該ビーズを通って試薬の拡散を可能にする細孔を含むことを特徴とする、核酸反応を行うためのビーズ。 - 前記ビーズが、約2μm~約120μmの直径を有する、請求項1記載のビーズ。
- 前記ヒドロゲルポリマーが、ポリエチレングリコール(PEG)‐チオール、PEG‐アクリレート、アクリルアミド、N,N'‐ビス(アクリロイル)シスタミン、PEG、ポリプロピレンオキシド(PPO)、ポリアクリル酸、ポリ(ヒドロキシエチルメタクリレート)(PHEMA)、ポリ(メチルメタクリレート)(PMMA)、ポリ(N‐イソプロピルアクリルアミド)(PNIPAAm)、ポリ(乳酸)(PLA)、乳酸‐グリコール酸共重合体(PLGA)、ポリカプロラクトン(PCL)、ポリ(ビニルスルホン酸)(PVSA)、ポリ(L‐アスパラギン酸)、ポリ(L‐グルタミン酸)、ポリリジン、寒天、アガロース、アルギネート、ヘパリン、硫酸化アルギン酸塩、硫酸デキストラン、ヒアルロナン、ペクチン、カラギーナン、ゼラチン、キトサン、セルロース、コラーゲン、ビスアクリルアミド、ジアクリレート、ジアリルアミン、トリアリルアミン、ジビニルスルホン、ジエチレングリコールジアリルエーテル、エチレングリコールジアクリレート、ポリメチレングリコールジアクリレート、ポリエチレングリコールジアクリレート、トリメチロールプロパントリメタクリレート、エトキシル化トリメチロールトリアクリレート、またはエトキシル化ペンタエリスリトールテトラアクリレート、或いはそれらの組合せまたは混合物を含む、請求項1または2記載のビーズ。
- 前記ヒドロゲルポリマーが、PEG‐チオール/PEG‐アクリレート、アクリルアミド/N,N'‐ビス(アクリロイル)シスタミン(BACy)、またはPEG/PPOを含む、請求項3記載のビーズ。
- 前記遺伝物質が、細胞、核酸、またはマイクロバイオームである、請求項1~4のいずれか1項記載のビーズ。
- 前記細胞が、哺乳動物細胞または細菌細胞である、請求項5記載のビーズ。
- 前記細胞が、Escherichia coli細胞、Bacillus subtilis細胞、Aeromonas hydrophila細胞、または線維芽細胞である、請求項5または6記載のビーズ。
- 前記核酸が、300塩基対以上のDNAまたはRNAである、請求項5記載のビーズ。
- 前記試薬が、50塩基対未満のサイズを有する酵素、化学物質およびプライマーを含む、請求項1~8のいずれか1項記載のビーズ。
- 前記試薬が、リゾチーム、プロテイナーゼK、ランダムヘキサマー、ポリメラーゼ(Φ29DNAポリメラーゼ、Taqポリメラーゼ、Bsuポリメラーゼ)、トランスポサーゼ(Tn5)、プライマー(P5およびP7アダプタ配列)、リガーゼ、触媒酵素、デオキシヌクレオチド三リン酸、緩衝剤または二価カチオンを含む、請求項1~9のいずれか1項記載のビーズ。
- 遺伝物質をヒドロゲルポリマーと溶液中で混合する工程、および
該溶液を不混和性流体と混合して、該遺伝物質をカプセル化するヒドロゲルビーズを形成する工程
を含み、
該ヒドロゲルビーズが、該遺伝物質を保持しながら、該ヒドロゲルビーズを通って試薬の拡散を可能にする細孔を含む、ヒドロゲルビーズ内に遺伝物質をカプセル化する方法。 - 前記ヒドロゲルビーズは、約2μm~約120μmの直径を有する、請求項11記載の方法。
- 前記ヒドロゲルポリマーが、ポリエチレングリコール(PEG)‐チオール、PEG‐アクリレート、アクリルアミド、N,N'‐ビス(アクリロイル)シスタミン(BACy)、PEG、ポリプロピレンオキシド(PPO)、ポリアクリル酸、ポリ(ヒドロキシエチルメタクリレート)(PHEMA)、ポリ(メチルメタクリレート)(PMMA)、ポリ(N‐イソプロピルアクリルアミド)(PNIPAAm)、ポリ(乳酸)(PLA)、乳酸‐グリコール酸共重合体(PLGA)、ポリカプロラクトン(PCL)、ポリ(ビニルスルホン酸)(PVSA)、ポリ(L‐アスパラギン酸)、ポリ(L‐グルタミン酸)、ポリリジン、寒天、アガロース、アルギネート、ヘパリン、硫酸化アルギン酸塩、硫酸デキストラン、ヒアルロナン、ペクチン、カラギーナン、ゼラチン、キトサン、セルロース、コラーゲン、ビスアクリルアミド、ジアクリレート、ジアリルアミン、トリアリルアミン、ジビニルスルホン、ジエチレングリコールジアリルエーテル、エチレングリコールジアクリレート、ポリメチレングリコールジアクリレート、ポリエチレングリコールジアクリレート、トリメチロールプロパントリメタクリレート、エトキシル化トリメチロールトリアクリレート、またはエトキシル化ペンタエリスリトールテトラアクリレート、或いはそれらの組合せまたは混合物を含む、請求項11または12記載の方法。
- 前記ヒドロゲルポリマーが、PEG‐チオール/PEG‐アクリレート、アクリルアミド/N,N'‐ビス(アクリロイル)シスタミン(BACy)、またはPEG/PPOを含む、請求項13記載の方法。
- 前記遺伝物質が、細胞、核酸、またはマイクロバイオームである、請求項11~14のいずれか1項記載の方法。
- 前記細胞が哺乳動物細胞または細菌細胞である、請求項15記載の方法。
- 前記細胞が、Escherichia coli細胞、Bacillus subtilis細胞、Aeromonas hydrophila細胞、または線維芽細胞である、請求項15または16記載の方法。
- 前記核酸が300塩基対以上のDNAまたはRNAである、請求項15記載の方法。
- 前記試薬が、50塩基対未満のサイズを有する酵素、化学物質、およびプライマーを含む、請求項11~18のいずれか1項記載の方法。
- 前記試薬が、リゾチーム、プロテイナーゼK、ランダムヘキサマー、ポリメラーゼ(Φ29DNAポリメラーゼ、Taqポリメラーゼ、Bsuポリメラーゼ)、トランスポサーゼ(Tn5)、プライマー(P5およびP7アダプタ配列)、リガーゼ、触媒酵素、デオキシヌクレオチド三リン酸、緩衝剤または二価カチオンを含む、請求項11~19のいずれか1項記載の方法。
- ヒドロゲルポリマーおよび遺伝物質を含む水溶液を不混和性流体と混合する工程、
該水溶液を液滴発生器に投入する工程、並びに
該遺伝物質をカプセル化するヒドロゲルビーズを生成する工程
を含み、
該ヒドロゲルビーズが、該遺伝物質を保持しながら、ヒドロゲルビーズを通る試薬の拡散を可能にする細孔を含むことを特徴とする、ヒドロゲルビーズ内に遺伝物質をカプセル化する方法。 - 前記ヒドロゲルビーズが、約2μm~約150μmの直径を有する、請求項21記載の方法。
- 前記ヒドロゲルポリマーがポリエチレングリコール(PEG)‐チオール/PEG‐アクリレート、アクリルアミド/N,N'‐ビス(アクリロイル)シスタミン(BACy)、PEG/ポリプロピレンオキシド(PPO)、ポリアクリル酸、ポリ(ヒドロキシエチルメタクリレート)(PHEMA)、ポリ(メチルメタクリレート)(PMMA)、ポリ(N‐イソプロピルアクリルアミド)(PNIPAAm)、ポリ(乳酸)(PLA)、乳酸‐グリコール酸共重合体(PLGA)、ポリカプロラクトン(PCL)、ポリ(ビニルスルホン酸)(PVSA)、ポリ(L‐アスパラギン酸)、ポリ(L‐グルタミン酸)、ポリリジン、寒天、アガロース、アルギネート、ヘパリン、硫酸化アルギン酸塩、硫酸デキストラン、ヒアルロナン、ペクチン、カラギーナン、ゼラチン、キトサン、セルロース、コラーゲン、ビスアクリルアミド、ジアクリレート、ジアリルアミン、トリアリルアミン、ジビニルスルホン、ジエチレングリコールジアリルエーテル、エチレングリコールジアクリレート、ポリメチレングリコールジアクリレート、ポリエチレングリコールジアクリレート、トリメチロールプロパントリメタクリレート、エトキシル化トリメチロールトリアクリレート、またはエトキシル化ペンタエリスリトールテトラアクリレート、或いはそれらの組合せまたは混合物を含む、請求項21または22記載の方法。
- 前記ヒドロゲルポリマーは、PEG‐チオール/PEG‐アクリレート、アクリルアミド/N,N'‐ビス(アクリロイル)シスタミン(BACy)、またはPEG/PPOを含む、請求項23記載方法。
- 前記遺伝物質が、細胞、核酸、またはマイクロバイオームである、請求項21~24のいずれか1項記載の方法。
- 前記細胞が哺乳動物細胞または細菌細胞である、請求項25記載の方法。
- 前記細胞が、Escherichia coli細胞、Bacillus subtilis細胞、Aeromonas hydrophila細胞、または線維芽細胞である、請求項25または26記載の方法。
- 前記核酸が300塩基対以上のDNAまたはRNAである、請求項25記載の方法。
- 前記試薬が、酵素、化学物質、および50塩基対未満のサイズを有するプライマーを含む、請求項21~28のいずれか1項記載の方法。
- 前記試薬が、リゾチーム、プロテイナーゼK、ランダムヘキサマー、ポリメラーゼ(Φ29DNAポリメラーゼ、Taqポリメラーゼ、Bsuポリメラーゼ)、トランスポサーゼ(Tn5)、プライマー(P5およびP7アダプタ配列)、リガーゼ、触媒酵素、デオキシヌクレオチド三リン酸、緩衝剤または二価カチオンを含む、請求項21~29のいずれか1項記載の方法。
- 請求項1~10のいずれか1項記載のビーズを得る工程、
ビーズ内にカプセル化された遺伝物質を増幅する工程、
ビーズ内にカプセル化された該遺伝物質に対してタグメンテーション反応を行う工程、および
該遺伝物質を配列決定し、それにより、ビーズ内にカプセル化された核酸ライブラリを生成する工程
を含むことを特徴とする、ビーズ内にカプセル化された遺伝物質から核酸ライブラリを調製する方法。 - 前記遺伝物質が細胞であり、更に該細胞を溶解して、核酸を抽出することを含む、請求項31に記載の方法。
- 前記細胞をリゾチームで溶解し、プロテイナーゼKで処理して核酸を抽出する、請求項32記載の方法。
- 前記タグメンテーション反応が、遺伝物質をアダプタ配列およびトランスポソームを含むトランスポサーゼ混合物と接触させることを含む、請求項31~33のいずれか1項記載の方法。
- 前記核酸ライブラリを固体支持体上に播種することを更に含む、請求項31~34のいずれか1項記載の方法。
- 前記播種が、前記ビーズを分解して、前記ビーズから前記核酸ライブラリを放出させることを含む、請求項35記載の方法。
- 前記ビーズは、前記ビーズを切断混合物と接触させることにより、または前記ビーズを約90℃に加熱して前記核酸ライブラリを放出させることによって分解される、請求項36記載の方法。
- 前記切断混合物が、ジチオスレイトール(DTT)、トリス(2‐カルボキシエチル)ホスフィン(TCEP)、またはトリス(3‐ヒドロキシプロピル)ホスフィン(THP)を含む、請求項37記載の方法。
- 前記固体支持体がフローセルデバイスである、請求項35~38のいずれか1項記載の方法。
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SG11201911871TA (en) | 2020-01-30 |
JP7032452B2 (ja) | 2022-03-08 |
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AU2022203968A1 (en) | 2022-06-30 |
AU2018312560B2 (en) | 2022-03-10 |
KR20200026218A (ko) | 2020-03-10 |
US20220411859A1 (en) | 2022-12-29 |
CA3067048C (en) | 2024-03-05 |
JP2022071023A (ja) | 2022-05-13 |
NZ759924A (en) | 2023-07-28 |
KR20210121294A (ko) | 2021-10-07 |
JP2020533950A (ja) | 2020-11-26 |
WO2019028166A1 (en) | 2019-02-07 |
KR102694654B1 (ko) | 2024-08-12 |
JP7282942B2 (ja) | 2023-05-29 |
KR102480894B1 (ko) | 2022-12-23 |
KR102307473B1 (ko) | 2021-10-01 |
AU2018312560A1 (en) | 2020-01-02 |
KR20230004943A (ko) | 2023-01-06 |
EP3662084A1 (en) | 2020-06-10 |
CN111094585A (zh) | 2020-05-01 |
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