JP2016513958A - ゲルパターン化した表面 - Google Patents
ゲルパターン化した表面 Download PDFInfo
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- JP2016513958A JP2016513958A JP2015559012A JP2015559012A JP2016513958A JP 2016513958 A JP2016513958 A JP 2016513958A JP 2015559012 A JP2015559012 A JP 2015559012A JP 2015559012 A JP2015559012 A JP 2015559012A JP 2016513958 A JP2016513958 A JP 2016513958A
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Abstract
Description
本出願は、2013年2月26日出願の米国仮特許出願第61/769289号および2013年3月6日出願の米国出願第13/787396号の利益を主張する。上記の出願はいずれも、その全体を本明細書に参照として組み込む。
この実施例は、シラン非含有アクリルアミド(SFA)でナノウェル基板をコーティングし、その後、チオホスフェートプライマーによりグラフト化し、ゲルグラフト化プライマーを相補的な蛍光オリゴヌクレオチドにハイブリダイズして、機能化手法の成功を裏付けることを実証している。
複数の技法を使用して、次いでゲル材料をローディングすることができる構造化アレイを製造できる。
この実施例は、ゲル含有ウェルのアレイの製造、ウェル中の核酸クラスターの増幅、およびクラスター中の核酸のシークエンシングを示す。
Claims (96)
- 表面を含む固体支持体であって、前記表面が複数のウェルを含み、前記ウェルがゲル材料を含有し、前記ウェルが表面上の間隙領域によって互いに分けられ、前記間隙領域が、各ウェルのゲル材料を、他の複数のウェルのゲル材料から隔離する、固体支持体と、
ゲル材料中の標的核酸のライブラリーであって、各ウェルのゲル材料がライブラリーの明白な単一種の標的核酸を含む、ライブラリーと
を含む、アレイ。 - 各ウェルの体積が最大1000μm3である、請求項1に記載のアレイ。
- 各ウェル中のゲルの体積が最大1000μm3である、請求項1に記載のアレイ。
- 各ウェルが、最大100μm2の開口部を表面に含む、請求項1に記載のアレイ。
- 複数のウェルが、繰り返しパターンを有するアレイを構成する、請求項1から4のいずれか一項に記載のアレイ。
- パターン中のウェルが、5マイクロメートル以下のピッチを有する、請求項5に記載のアレイ。
- 複数のウェルが、ランダムなパターンを有するアレイを構成する、請求項1から4のいずれか一項に記載のアレイ。
- ゲルが、ヒドロゲルを含む、請求項1から7のいずれか一項に記載のアレイ。
- ゲルが、SFAまたはPAZAMを含む、請求項1から7のいずれか一項に記載のアレイ。
- 各ウェル中のゲル材料が、ライブラリー中の単一種の標的核酸を含む、請求項1から9のいずれか一項に記載のアレイ。
- 単一種の複数のコピーが、各ウェル中で核酸分子のクローン集団を構成する、請求項10に記載のアレイ。
- 単一種の複数のコピーが、核酸分子中でコンカテマー反復として存在する、請求項10に記載のアレイ。
- 単一種が、単なる単一核酸分子として、各ウェル中に存在する、請求項1から10のいずれか一項に記載のアレイ。
- 単一核酸分子が、2本の相補鎖を含む、請求項13に記載のアレイ。
- 表面が、少なくとも1000個のウェル/mm2の密度を含む、請求項1から13のいずれか一項に記載のアレイ。
- ゲル材料が、ウェルの表面に共有結合する、請求項1から15のいずれか一項に記載のアレイ。
- (a)平面を含む固体支持体を設けるステップであって、前記平面が1つ以上の凹状フィーチャーによって断続され、前記1つ以上の凹状フィーチャーが、平面上の1つ以上の間隙領域に隣接するステップと、
(b)固体支持体の少なくとも一部をゲル材料でコーティングするステップであって、前記一部が少なくとも1つの凹状フィーチャーおよび少なくとも1つの間隙領域を含むステップと、
(c)平面を研磨し、ゲル材料を少なくとも1つの間隙領域から除去し、少なくとも1つの凹状フィーチャー中にゲル材料を保持するステップと
を含む、基板を作製する方法。 - 凹状フィーチャーが、間隙領域に取り囲まれるウェルを含む、請求項17に記載の方法。
- 各ウェルの体積が最大1000μm3である、請求項18に記載の方法。
- 各ウェル中のゲルの体積が最大1000μm3である、請求項18に記載の方法。
- 各ウェルが、表面中に最大100μm2の開口部を有する開口部を含む、請求項18に記載の方法。
- ウェルが、繰り返しパターンを有するアレイを構成する、請求項18から21のいずれか一項に記載の方法。
- パターン中のウェルが、5μm以下のピッチを有する、請求項22に記載の方法。
- 複数のウェルが、ランダムなパターンを有するアレイを構成する、請求項18から22のいずれか一項に記載の方法。
- 表面が、少なくとも1000個のウェル/mm2の密度を含む、請求項18から24のいずれか一項に記載の方法。
- コーティングが、固体支持体を、予め生成しておいたゲル材料と接触させることを含む、請求項17から24のいずれか一項に記載の方法。
- コーティングが、固体支持体を、次いでゲル材料を生成する液体と接触させることを含む、請求項17から24のいずれか一項に記載の方法。
- ゲル材料が、ヒドロゲルを含む、請求項17から27のいずれか一項に記載の方法。
- ゲル材料が、SFAまたはPAZAMを含む、請求項17から27のいずれか一項に記載の方法。
- ゲル材料が、核酸にさらに付着されている、請求項17から29のいずれか一項に記載の方法。
- 核酸が、増幅用プライマーを含む、請求項30に記載の方法。
- 核酸が、鋳型核酸を含み、方法が、凹状フィーチャー中のゲル材料中の鋳型核酸を増幅することをさらに含む、請求項30または31に記載の方法。
- 増幅が、鋳型核酸のコピーを複数作製し、核酸分子のクローン集団を形成する、請求項32に記載の方法。
- 増幅が、鋳型核酸のコピーを複数作製して、核酸分子のコンカテマー反復を形成する、請求項32に記載の方法。
- ゲル材料をウェルの表面に共有結合させることをさらに含む、請求項18から34のいずれか一項に記載の方法。
- 研磨が、ビーズのスラリーによる機械的な摩耗を含む、請求項17から35のいずれか一項に記載の方法。
- 研磨が、固体支持体の表面を拭く、または擦ることによる機械的な摩耗を含む、請求項17から36のいずれか一項に記載の方法。
- 研磨が、化学的研磨を含む、請求項17から37のいずれか一項に記載の方法。
- ゲル材料をタンパク質にさらに付着させる、請求項17から38のいずれか一項に記載の方法。
- (a)表面を含む固体支持体を設けるステップであって、前記表面が複数のウェルを含み、前記ウェルがゲル材料を含有し、前記ウェルが表面上の間隙領域によって互いに分けられ、前記間隙領域が、各ウェルのゲル材料を、他の複数のウェルのゲル材料から隔離する、ステップと、
(b)標的核酸のライブラリーを固体支持体のウェルに送達して、各ウェル中のゲル材料に付着している単一種の標的核酸を有するウェルのアレイを製造するステップであって、アレイ中の種々のウェルが、ライブラリーからの種々の標的核酸種を含むステップと、
(c)アレイのウェル中のゲル材料に付着している標的核酸を増幅して、アレイの各ウェルで個々の標的核酸のクローン集団を生成するステップと
を含む、アレイを作製する方法。 - 各ウェル中のゲルの体積が最大1000μm3である、請求項40に記載の方法。
- 各ウェルが、表面中に最大100μm2の開口部を有する開口部を含む、請求項40に記載の方法。
- 複数のウェルが、表面上に繰り返しパターンを構成する、請求項40に記載の方法。
- パターン中のウェルが、5μm以下のピッチを有する、請求項43に記載の方法。
- 複数のウェルが、表面上にランダムなパターンを構成する、請求項40に記載の方法。
- ゲルがヒドロゲルを含む、請求項40に記載の方法。
- ゲルがSFAまたはPAZAMを含む、請求項40に記載の方法。
- 表面が、少なくとも1000個のウェル/mm2の密度を含む、請求項40に記載の方法。
- ゲル材料をウェルの表面に共有結合させる、請求項40に記載の方法。
- ステップ(a)における固体支持体を設けることが、
(i)平面を含む固体支持体を設けることであって、前記表面が複数のウェルを含み、前記ウェルが表面上の間隙領域によって互いに分けられることと、
(ii)前記ウェルおよび前記間隙領域をゲル材料でコーティングすることと、
(iii)平面を研磨して、少なくとも1つの間隙領域からゲル材料を除去し、ウェル中のゲル材料を保持することと
を含む、請求項40から49のいずれか一項に記載の方法。 - コーティングが、固体支持体を、予め生成しておいたゲル材料と接触させることを含む、請求項50に記載の方法。
- コーティングが、固体支持体を、次いでゲル材料を生成する液体と接触させることを含む、請求項50または51に記載の方法。
- 研磨が、ビーズのスラリーによる機械的な摩耗を含む、請求項50から52のいずれか一項に記載の方法。
- 研磨が、固体支持体の表面を拭く、または擦ることによる機械的な摩耗を含む、請求項50から53のいずれか一項に記載の方法。
- 研磨が、化学的研磨を含む、請求項50から54のいずれか一項に記載の方法。
- ステップ(b)がステップ(c)の前に行われる、請求項40から55のいずれか一項に記載の方法。
- ステップ(b)およびステップ(c)が同時に行われる、請求項40から55のいずれか一項に記載の方法。
- ステップ(b)の送達が、ライブラリーを、流体中の標的核酸混合液として準備し、前記流体を固体支持体に接触させ、それによって、標的核酸が、ウェルおよび間隙領域への流体アクセスを有することを含む、請求項40から57のいずれか一項に記載の方法。
- 増幅が、固相ポリメラーゼ連鎖反応(PCR)、多置換増幅(MDA)またはローリングサークル増幅(RCA)を含む、請求項40から58のいずれか一項に記載の方法。
- 増幅が、ウェル中のゲル材料に付着している少なくとも1つのプライマー種のポリメラーゼ伸長を含む、請求項59に記載の方法。
- 増幅が、ウェル中のゲル材料に付着している少なくとも2つのプライマー種を使用したブリッジ増幅を含む、請求項40から58のいずれか一項に記載の方法。
- クローン集団がそれぞれ、ライブラリーからの少なくとも1000個の標的核酸のコピーを含む、請求項40から61のいずれか一項に記載の方法。
- (a)表面および核酸ライブラリーを含む固体支持体を設けるステップであって、前記表面が複数のウェルを含み、前記ウェルがゲル材料を含有し、前記ウェルが、表面上の間隙領域によって互いに分けられ、前記間隙領域が、各ウェルのゲル材料を、他の複数のウェルのゲル材料から隔離し、ライブラリーの単一種の標的核酸が、各ウェルのゲル材料に付着している、ステップと、
(b)固体支持体を、標的核酸に結合する少なくとも1つのプローブと接触させるステップと、
(c)固体支持体を検出して、少なくとも1つのプローブに結合する標的核酸種を有するウェルを識別するステップと
を含む、核酸を検出する方法。 - 各ウェルのゲルの体積が、最大1000μm3である、請求項63に記載の方法。
- 各ウェルが、表面中に最大100μm2の開口部を有する開口部を含む、請求項63に記載の方法。
- 複数のウェルが、表面上に繰り返しパターンを構成する、請求項63に記載の方法。
- パターン中のウェルが、5マイクロメートル以下のピッチを有する、請求項66に記載の方法。
- 複数のウェルが、表面上にランダムなパターンを構成する、請求項63に記載の方法。
- ゲルがヒドロゲルを含む、請求項63に記載の方法。
- ゲルがSFAまたはPAZAMを含む、請求項63に記載の方法。
- 表面が、少なくとも1000個のウェル/mm2の密度を含む、請求項63に記載の方法。
- ゲル材料が、ウェルの表面に共有結合する、請求項63に記載の方法。
- ステップ(a)における固体支持体を設けることが、
(i)平面を含む固体支持体を設けることであって、前記表面が複数のウェルを含み、前記ウェルが表面上の間隙領域によって互いに分けられることと、
(ii)前記ウェルおよび前記間隙領域をゲル材料でコーティングすることと、
(iii)平面を研磨して、少なくとも1つの間隙領域からゲル材料を除去し、ウェル中のゲル材料を保持することと
を含む、請求項63から72のいずれか一項に記載の方法。 - コーティングが、固体支持体を、予め生成しておいたゲル材料と接触させることを含む、請求項73に記載の方法。
- コーティングが、固体支持体を、次いでゲル材料を生成する液体と接触させることを含む、請求項73または74に記載の方法。
- 研磨が、ビーズのスラリーによる機械的な摩耗を含む、請求項73から75のいずれか一項に記載の方法。
- 研磨が、固体支持体の表面を拭く、または擦ることによる機械的な摩耗を含む、請求項73から76のいずれか一項に記載の方法。
- 研磨が、化学的研磨を含む、請求項73から77のいずれか一項に記載の方法。
- ステップ(a)における設けることが、
(iv)ライブラリーの単一種の標的核酸を各ウェルのゲル材料に付着させる条件下で、標的核酸のライブラリーを固体支持体のウェルに送達することと、
(v)アレイのウェル中のゲル材料に付着している標的核酸を増幅して、アレイの各ウェルで個々の標的核酸のクローン集団を生成することと
をさらに含む、請求項73から77のいずれか一項に記載の方法。 - ステップ(iv)がステップ(v)の前に行われる、請求項79に記載の方法。
- ステップ(iv)およびステップ(v)が同時に行われる、請求項79に記載の方法。
- ステップ(iv)における送達が、ライブラリーを、流体中の標的核酸混合液として準備し、前記流体を固体支持体に接触させ、それによって、標的核酸が、ウェルおよび間隙領域への流体アクセスを有することを含む、請求項79から81のいずれか一項に記載の方法。
- 増幅が、固相ポリメラーゼ連鎖反応(PCR)、多置換増幅(MDA)またはローリングサークル増幅(RCA)を含む、請求項79から82のいずれか一項に記載の方法。
- 増幅が、ウェル中のゲル材料に付着している少なくとも1つのプライマー種を使用する、請求項83に記載の方法。
- 増幅が、ウェル中のゲル材料に付着している少なくとも2つのプライマー種を使用したブリッジ増幅を含む、請求項79から82のいずれか一項に記載の方法。
- クローン集団がそれぞれ、ライブラリーからの少なくとも1000個の標的核酸のコピーを含む、請求項79から85のいずれか一項に記載の方法。
- 少なくとも1つのプローブが、少なくとも1種の標的核酸の少なくとも一部に相補的な少なくとも1種の核酸を含む、請求項63から86のいずれか一項に記載の方法。
- 少なくとも1つのプローブが、ステップ(c)で検出される蛍光標識を含む、請求項87に記載の方法。
- 少なくとも1つのプローブが、ポリメラーゼおよびヌクレオチドをさらに含む、請求項87に記載の方法。
- ステップ(c)における検出が、ヌクレオチドのプローブ中への、または標的核酸中への取り込みを検出することを含む、請求項89に記載の方法。
- ステップ(c)における検出が、合成時解読法を含む、請求項89に記載の方法。
- シークエンシング法において、ステップ(b)および(c)を数回繰り返す、請求項63から91のいずれか一項に記載の方法。
- プローブが、タンパク質と結合する核酸を含む、請求項63から91のいずれか一項に記載の方法。
- 検出が、光学的検出、電気的検出、および熱検出からなる群から選択される、請求項63から93のいずれか一項に記載の方法。
- 光学的検出が、蛍光、ルミネセンス、化学ルミネセンス、吸光度、および蛍光共鳴エネルギー移動からなる群から選択される、請求項94に記載の方法。
- (a)平面を含む固体支持体を設けるステップであって、前記平面が、1つ以上の凹状フィーチャーによって断続され、前記凹状フィーチャーがゲル材料を含有し、1つ以上の凹状フィーチャーが、平面上の1つ以上の間隙領域に隣接し、前記間隙領域が実質的にゲル材料を含まず、前記ゲル材料が標的検体を含む、ステップと、
(b)標的検体が特異的にプローブと相互作用する条件下で、前記固体支持体をプローブに接触させるステップと、
(c)前記固体支持体を検出して、プローブの1つ以上と相互作用する標的検体の少なくとも1つのサブセットを識別するステップと
を含む、検体を検出する方法。
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