JP6959330B2 - 核酸増幅方法および核酸解析用装置 - Google Patents
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Description
任意の規則性を持って配置された親水性を有する複数の第一の表面と、前記複数の第一の表面のそれぞれを囲む、前記第一の表面より親水性が低い第二の表面とを有する基板であって、前記第一の表面には鋳型となる核酸と特異的に結合する分子が固定または配置される、基板を準備する工程と、
前記基板上に、前記鋳型となる核酸を含む試料溶液と核酸増幅基質および核酸合成酵素を含む反応溶液との混合溶液を供給し、前記第一の表面に前記混合溶液が配置され、前記鋳型となる核酸と特異的に結合する分子に前記鋳型となる核酸が結合する工程と、
前記基板上に疎水性溶媒を供給し、前記第一の表面上に配置された前記混合溶液が封入された液滴を形成する工程と、
前記液滴内で前記核酸の増幅反応を行う工程と、
前記基板上から前記疎水性溶媒を除去する工程と、
前記基板上に核酸増幅基質および核酸合成酵素を含む反応溶液を供給する工程と、
前記核酸の増幅反応を行う工程と
を含むことを特徴とする核酸増幅方法を提供する。
本実施例における基板上核酸増幅方法の各工程について、図6を用いて説明する。
101…親水性パターン領域
102…基板
103…フローセル溶液槽
104…第一の開口部
105…第二の開口部
106…試料溶液槽
107…疎水性溶媒槽
108…反応溶液槽
109…排液槽
110…バルブ
112…ポンプ
201…下層基板
202…隔壁
203…鋳型核酸を固定するための分子
305…液滴
308…増幅核酸断片
310…試料溶液
320…疎水性溶媒
330…反応溶液
501…増幅核酸断片を持つ親水性パターン領域
840…試料溶液と反応溶液との混合溶液
913…溶液混合槽
914…洗浄溶液槽
915、916…温度制御装置
917…観察部
918…制御部
1301…基板上核酸クラスター形成試薬セット
1302…DNAシーケンス試薬セット
Claims (12)
- 親水性を有する複数の第一の表面と、前記複数の第一の表面のそれぞれを囲む、前記第一の表面より親水性が低い第二の表面とを有する基板であって、前記第一の表面には鋳型となる核酸と特異的に結合する分子が固定される、基板を準備する工程と、
前記基板上に、前記鋳型となる核酸を含む試料溶液と核酸増幅基質および核酸合成酵素を含む反応溶液との混合溶液を供給し、前記第一の表面に前記混合溶液が配置され、前記鋳型となる核酸と特異的に結合する分子に前記鋳型となる核酸が結合する工程と、
前記基板上に疎水性溶媒を供給し、前記第一の表面上に配置された前記混合溶液が封入された液滴を形成する工程と、
前記液滴内で前記核酸の増幅反応を行う工程と、
前記基板上から前記疎水性溶媒を除去する工程と、
前記基板上に核酸増幅基質および核酸合成酵素を含む反応溶液を供給する工程と、
前記核酸の増幅反応を行う工程と
を含むことを特徴とする核酸増幅方法。 - 前記増幅反応はローリングサークル増幅(RCA)またはポリメラーゼ連鎖反応(PCR)である、請求項1に記載の核酸増幅方法。
- 前記鋳型となる核酸を含む試料溶液を希釈して、前記液滴1つ当たり1分子以下の前記鋳型となる核酸が封入された液滴を形成する、請求項1又は2に記載の核酸増幅方法。
- 前記核酸は、メッセンジャーRNA(mRNA)、非コードRNA(ncRNA)、マイクロRNA、ゲノムDNA、およびそれらの断片、ならびにRNAとDNAのハイブリッド核酸からなる群より選択される、請求項1〜3のいずれか1項に記載の核酸増幅方法。
- 前記液滴内で前記核酸の増幅反応を行う工程の後、
前記基板上に、前記鋳型となる核酸を含む試料溶液と核酸増幅基質および核酸合成酵素を含む反応溶液との混合溶液を供給し、前記第一の表面のうち増幅された核酸断片を含まない第一の表面に前記混合溶液が配置され、前記鋳型となる核酸と特異的に結合する分子に前記鋳型となる核酸が結合する工程と、
前記基板上に疎水性溶媒を供給し、前記第一の表面上に配置された前記混合溶液が封入された液滴を形成する工程と、
前記液滴内で前記核酸の増幅反応を行う工程と
をさらに含む、請求項1〜4のいずれか1項に記載の核酸増幅方法。 - 前記基板上から前記疎水性溶媒を除去する工程の後、前記基板上を乾燥する工程をさらに含む、請求項1〜5のいずれか1項に記載の核酸増幅方法。
- 核酸増幅反応を行う反応容器と、
鋳型となる核酸を含む試料溶液を収容する試料溶液槽と、
疎水性溶媒を収容する疎水性溶媒槽と、
少なくとも核酸増幅基質を含む反応溶液を収容する反応溶液槽と
制御部と
を備えた核酸解析用装置であって、
前記反応容器には、前記試料溶液槽、前記疎水性溶媒槽、および前記反応溶液槽と連結可能な、第一の開口部と第二の開口部が設けられ、
前記反応容器内の上面または底面のいずれかに、親水性を有する複数の第一の表面と、前記複数の第一の表面のそれぞれを囲む、前記第一の表面より親水性が低い第二の表面とを有する基板が設けられており、
前記第一の表面には、鋳型となる核酸と特異的に結合する分子が固定され、
前記制御部は、請求項1〜6のいずれか1項に記載の方法を実施するように前記核酸解析用装置を制御することを特徴とする前記核酸解析用装置。 - 前記基板の形状は凹構造を有しており、前記凹構造の最底面が前記第一の表面である、請求項7に記載の核酸解析用装置。
- 前記第一の表面は直径0.5〜2.0μmの大きさであり、前記基板上における前記第一の表面の密度は20万個/mm2以上である、請求項7又は8に記載の核酸解析用装置。
- 前記基板上を観察可能な観察部、および/または
前記反応容器の排液を回収する排液槽
をさらに備える、請求項7〜9のいずれか1項に記載の核酸解析用装置。 - 少なくとも1つの温度制御装置をさらに備える、請求項7〜10のいずれか1項に記載の核酸解析用装置。
- 前記反応容器、前記試料溶液槽、前記疎水性溶媒槽、および前記反応溶液槽が、前記温度制御装置により適切な温度に調整可能である、請求項11に記載の核酸解析用装置。
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EP2776165A2 (en) * | 2011-11-07 | 2014-09-17 | Illumina, Inc. | Integrated sequencing apparatuses and methods of use |
US8895249B2 (en) | 2012-06-15 | 2014-11-25 | Illumina, Inc. | Kinetic exclusion amplification of nucleic acid libraries |
JP5651643B2 (ja) * | 2012-07-23 | 2015-01-14 | 株式会社日立ハイテクノロジーズ | 核酸分析方法 |
US9914968B2 (en) * | 2012-09-26 | 2018-03-13 | Cepheid | Honeycomb tube |
CN103849674B (zh) * | 2012-11-30 | 2015-12-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | 基于亲疏水模式基片的微液相反应方法 |
US20160199832A1 (en) * | 2013-08-30 | 2016-07-14 | Advanced Liquid Logic France Sas | Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces |
EP3080300B1 (en) * | 2013-12-11 | 2020-09-02 | Genapsys Inc. | Systems and methods for biological analysis and computation |
KR102437408B1 (ko) | 2014-07-08 | 2022-08-29 | 고쿠리츠켄큐카이하츠호진 카가쿠기쥬츠신코키코 | 물질 봉입 방법 및 타깃 분자를 검출하는 방법 |
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2017
- 2017-04-05 CN CN201780089341.2A patent/CN110520529A/zh active Pending
- 2017-04-05 WO PCT/JP2017/014213 patent/WO2018185871A1/ja unknown
- 2017-04-05 JP JP2019510554A patent/JP6959330B2/ja active Active
- 2017-04-05 EP EP17904384.9A patent/EP3608405A4/en not_active Withdrawn
- 2017-04-05 US US16/499,766 patent/US20200102587A1/en not_active Abandoned
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US20200102587A1 (en) | 2020-04-02 |
WO2018185871A1 (ja) | 2018-10-11 |
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EP3608405A1 (en) | 2020-02-12 |
EP3608405A4 (en) | 2020-10-28 |
JPWO2018185871A1 (ja) | 2019-12-19 |
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