JP2010178744A - ビーズエマルジョン核酸増幅 - Google Patents
ビーズエマルジョン核酸増幅 Download PDFInfo
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- JP2010178744A JP2010178744A JP2010053827A JP2010053827A JP2010178744A JP 2010178744 A JP2010178744 A JP 2010178744A JP 2010053827 A JP2010053827 A JP 2010053827A JP 2010053827 A JP2010053827 A JP 2010053827A JP 2010178744 A JP2010178744 A JP 2010178744A
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Abstract
【解決手段】油中水型エマルジョンを形成して、核酸鋳型、ビーズおよび増幅反応溶液が乳化された複数の水性マイクロリアクターを作製する段階;前記マイクロリアクター中で核酸を増幅してコピーを形成する段階;増幅されたコピーを前記マイクロリアクター中の前記ビーズに結合させる段階、を含む核酸の増幅方法。
【選択図】なし
Description
本発明は、ビーズのような固体支持体上で、少ないコピー数の核酸鋳型を、配列決定に適した量まで増幅する方法に関する。また本発明では、ゼロビーズ除去にも関し- 増幅した核酸を含む固体支持体を濃縮する方法も、開示される。
本願は、2003年1月29日付のUSSN 60/443,471、2003年4月23日付のUSSN 60/465,071、2003年6月6日付のUSSN 60/476,313、2003年6月6日付のUSSN 60/476,504、2003年6月6日付のUSSN 60/476,592、2003年6月6日付のUSSN 60/476,602、2003年8月25日付のUSSN 60/497,985に基づく優先権を伴う出願である。本段落中の全ての特許および特許出願は、参照として本明細書に組み入れられる。
現在の配列決定法の効率が悪いことを考慮すると、ゲノムライブラリーまたはcDNAライブラリーのような、多数の核酸配列を増幅できるということは、非常に重要である。現在の配列決定技術では、1回の配列決定反応あたり何百万もの核酸のコピーが必要である。さらに、ヒトゲノムの配列決定には、何千万もの異なる配列決定反応が必要である。開始材料が制限される場合、ゲノム配列決定の前に、最初のDNAの増幅が必要である。例えば、配列決定するゲノムがわずかな量の病原体または出生前の患者に由来する場合に、開始材料は制限されうる。現在のインビトロゲノム増幅技術では、面倒なクローニングおよび培養プロトコールが必要であり、そのためにゲノム配列決定の有用性が制限されてきた。PCRのような他の技術は迅速で信頼性が高いものの、代表的な様式ではゲノムを増幅できない。
ビーズエマルジョン増幅の簡単な概要
本発明の1つの態様の簡単な概要が以下に述べられている。この態様の各段階のより詳細な記述は、その後に述べられている。本態様では、PCRが、選択された増幅技術である。
1つの好ましい態様では、ビーズエマルジョン増幅によって増幅される核酸鋳型は、例えば、ゲノムDNAライブラリーまたはcDNAライブラリーのような、DNA集団である。DNA集団の各メンバーが、第1の末端で1つの共通の核酸配列を有し、かつ第2の末端で1つの共通の核酸配列を有することが好ましい。これは、例えば、DNA集団の各メンバーの片方の末端に第1のアダプターDNA配列を連結し、第2の末端に第2のアダプターDNA配列を連結することによって、実施できる。多くのDNAおよびcDNAライブラリーは、クローニングベクターの性質により(例えば、Bluescript、Stratagene、ラホヤ、カリフォルニア州)、各メンバーDNAが第1の末端に1つの共通配列、第2の末端に第2の共通配列を有するというこの記述に当てはまる。核酸鋳型は、インビトロ増幅(PCRおよび非対称PCRという好ましい増幅技術を含む)に適した任意のサイズで良い。1つの好ましい態様では、鋳型は、例えば約250 bpのような、約150bp〜750 bpのサイズである。
本発明の1つの局面では、増幅される一本鎖核酸鋳型が、捕捉ビーズに結合される。鋳型は、乳化の前、または乳化の後に、ビーズに捕捉される。1つの好ましい局面では、核酸鋳型の増幅コピーは、捕捉ビーズに結合される。非限定的実施例では、これらの結合は、ビーズの表面に結合する化学基またはオリゴヌクレオチドによって媒介される場合がある。核酸(例えば、核酸鋳型、増幅コピー、またはオリゴヌクレオチド)は、当技術分野で周知の任意の方法で、固体支持体(例えば、捕捉ビーズ)に結合できる。
本発明で使用するために、核酸鋳型の結合したまたはしていない捕捉ビーズは、熱安定性の油中水エマルジョンに懸濁される。複数のマイクロリアクターは、1つの鋳型および1つのビーズのみを含むと考えられる。鋳型を含まないまたはビーズを含まない液滴が多くあり得る。同様に、鋳型を複数コピー含む液滴もあり得る。エマルジョンは、当技術分野で周知の任意の適当な方法で形成できる。エマルジョンを作製する1つの方法が以下に記述されるが、エマルジョンを作製する任意の方法が使用できる。これらの方法は、当技術分野で周知であり、アジュバント法、カウンタフロー法(counter-flow method)、クロスカレント法(cross-current method)、回転ドラム法、およびメンブレン法を含む。さらに、マイクロカプセルのサイズは、流速および成分のスピードを変えることにより、調節できる。例えば、滴加では、液滴のサイズおよび総送達時間を変更することができる。好ましくは、エマルジョンには1マイクロリットルあたり約3,000の密度でビーズが封入されている。
封入後、ビーズに結合したまたはしていない鋳型核酸は、転写ベースの増幅系を含む任意の適当な増幅方法によって、増幅できる
。ジオリゴヌクレオチド増幅、等温増幅(isothermal amplification)(Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A) 89:392-396 (1992))、核酸配列ベースの増幅(Nucleic Acid Sequence Based Amplification)(NASBA; 例えばDeiman B et al., 2002, Mol Biotechnol. 20(2):163-79を参照されたい)、全ゲノム増幅(例えば、Hawkins TL et al., 2002, Curr Opin Biotechnol. 13(1):65-7を参照されたい)、鎖置換増幅(SDA)(例えば、Andras SC, 2001, Mol Biotechnol. 19(1):29-44を参照されたい)、ローリングサークル増幅(USP 5,714,320に概説)、および他の周知の技術のような他の方法も、本発明に従って使用できる。
PCR増幅のような増幅のための核酸プライマーの選択は、当業者の能力の範囲内である。プライマー設計の選択は、科学論文、例えば、Rubin, E. and A.A. Levy, Nucleic Acids Res, 1996. 24(18):p. 3538-45; およびBuck, G.A., et al., Biotechniques, 1999. 27(3): p. 528-36に記載されている。好ましい態様では、プライマーは2連のPCR/配列決定プライマーの効率的な合成のために、20塩基(5つのテトラマー)に限定され得る。各プライマーは、5'末端に2塩基GCクランプ、3'末端に単一のGCクランプを含む可能性があり、全てのプライマーは類似したTm(+/- 2℃)を有する。好ましい態様では、任意の設計されたプライマー内において、プライマー内のヘアピン構造(内部ヘアピンステム(internal hairpin stems)ΔG>-1.9 kcal/mol)がないことが強く推奨される。別の好ましい態様では、プライマーの2量化も制御され;最大で3塩基のダイマーが許容される。しかし、これは3'末端の最後の6つの塩基でのみ許容され、3'ダイマーの最大許容ΔGは、-2.0 kcal/molである。好ましくは、3'末端がグループ内の他の分子と類似し過ぎているプライマーには、ペナルティが適用される。これにより、1つのプライマーと、別のプライマーの逆相補分子の間のクロスハイブリダイゼーションが避けられる。
核酸鋳型の増幅および増幅されたコピーのビーズへの結合の後、エマルジョンは「破壊」(当技術分野では「解乳化」とも呼ぶ)される。エマルジョンの破壊には多くの方法があり(例えば、USP 5,989,892およびその引用文献を参照されたい)、当業者は、適当な方法を選択できるだろう。本発明では、エマルジョンを破壊する1つの好ましい方法は、さらなる油を用いて、エマルジョンを2つの相に分離することである。その後、油相を除去し、適当な有機溶媒(例えば、ヘキサン)を添加する。混合後、油/有機溶媒相を除去する。この段階は、数回繰り返すことができる。最後に、ビーズの上の水層を除去する。その後、有機溶媒およびアニーリング緩衝液(例えば、1つの適当なアニーリング緩衝液は、実施例に記述されている)を用いてビーズを洗浄し、その後、再度アニーリング緩衝液中で洗浄する。適当な有機溶媒には、メタノール、エタノール等のアルコールが含まれる。
この段階で、ビーズ上の増幅核酸は、ビーズ上で直接または異なる反応容器中で、配列決定できる。本発明の1つの態様では、ビーズを反応容器に移し、核酸を配列決定反応(例えば、ピロリン酸またはサンガーの配列決定)にかけることによって、核酸はビーズ上で直接配列決定される。または、ビーズを単離し、各ビーズから核酸を除去して配列決定することもできる。いずれの場合も、配列決定段階は、各々のビーズ上で行なう。しかし、この方法は商業的に実行可能で技術的にも可能であるものの、ビーズの多くは「陰性の」ビーズ(すなわち、増幅核酸の結合していないビーズ)であるため、あまり有効ではない可能性がある。そのような場合には、以下に概説する任意の過程を用いて、マルチウェル(例えば、ピコタイター)プレートに配分する前に、陰性のビーズを除去できる。
R(N) = exp - (N/M) x (N/M)N/N!(xはかけ算の符号)
1 - (0.005/0.09) = 94%
であるようなビーズの集団が残る。
鋳型DNAの捕捉、DNA増幅、および増幅された鋳型に結合したビーズの回収を含む以下の手順は、単一のチューブ中で実行できる。エマルジョンの形であるため、単一のチューブ中でビーズが100〜200μmのマイクロリアクターに物理的に分離し、様々な鋳型がクローン増幅できる。増幅産物の固定は、DNA捕捉ビーズに結合したオリゴヌクレオチドに沿って鋳型を伸長することで行なわれる。典型的には、固定された鋳型のコピー数は、ビーズ1つあたり1000万から3000万コピーの範囲である。単一種の核酸鋳型が複数コピー結合したDNA捕捉ビーズは、PTP上に分配できる。
本実施例は、1つの唯一の核酸鋳型が選択的に結合したビーズ集団の調製を記述する。クローン増幅の成功は、各ビーズに制御された数の鋳型種(0.5から1)を送達することに依存する。過剰な鋳型種が送達されると、混合した鋳型集団のPCR増幅が行われ、意味のある配列データが得られなくなり、鋳型種の数が足りないと、配列決定するための鋳型を含むウェルの数が減少してしまう。これにより、配列決定段階によって提供されるゲノムのカバー範囲が低下する。その結果、定量を繰り返して正確に鋳型濃度を決定し、以下に概説する結合プロトコールに従うことが好ましい。
エマルジョンPCR反応の成功は、鋳型種の質に関連している。増幅段階をいくら詳細に注意深く行なっても、鋳型の質が悪ければ、増幅の成功と意味のある配列データの生成が損なわれてしまう。時間と費用の無駄を避けるためには、本過程のエマルジョンPCR段階を開始する前に、鋳型材料の質を確認することが重要である。好ましくは、ライブラリーはエマルジョンPCRに使用する前に、2つの品質管理段階に合格する必要がある。その濃度および産物の分布を決定する必要がある。理想的には、ライブラリーはアダプターダイマー(例えば、〜90塩基)がほとんどまたは全く見られない異質な断片集団である。また、PCRプライマーを用いた増幅では、例えば、300から500 bpの範囲の産物のスミアが得られる。増幅産物がないということは、アダプターが鋳型に適切に連結されていないことを反映する可能性があり、サイズを問わず単一のバンドが存在することは鋳型が汚染されていたことを反映する可能性がある。
この段階で主に考慮するのは、散在するアンプリコンでPCR反応混合物を汚染しないようにすることである。残存アンプリコンによるPCR反応液の汚染は、配列決定段階の失敗に至る重要な問題の1つである。汚染の可能性を低下させるために、適切な実験室テクニックを用い、反応液の調製はクリーンルームのUV処理した層流フード内で実施する。
200μlのPCR反応混合物(600,000ビーズの増幅に充分な量)には、0.2 mlのPCRチューブ内で、以下の試薬を混合する。
1. 600,000個のDNA捕捉ビーズをストックチューブから1.5 ml遠心チューブへ移した。使用される量は、調製された試薬のビーズ濃度に依存する。
2. ベンチトップミニ遠心機でビーズを沈殿させ、上清を除去した。
3. 段階4〜11は、PCRクリーンルーム内で行った。
4. ビーズを1 mLの1Xアニーリング緩衝液で洗浄した。
5. マイクロ遠心機で捕捉ビーズを沈殿させた。チューブを180°回転させ、再度遠心した。
6. ビーズを含むチューブから上清を約10μl残して除去した。ビーズは乱さなかった。
7. 1 mLの1Xアニーリング緩衝液を添加し、混合液を1分間インキュベートした。その後、ビーズを段階5と同様にして沈殿させた。
8. チューブから内容物を約100μL残して除去した。
9. 残りのビーズと溶液をPCRチューブに移し替えた。
10. 150μLの1Xアニーリング緩衝液を用いて、数回ピペットで出し入れして、1.5 mLチューブを洗浄した。この液を、ビーズを含むPCRチューブに添加した。
11. 段階5と同様にしてビーズを沈殿させ、ビーズ沈殿を乱さないように注意しながら、上清を10μL残して除去した。
12. 定量した一本鎖鋳型DNA(sstDNA)の一部を除去した。最終濃度は200,000-sst DNA分子/μlだった。
13. 希釈したsstDNA 3μlをビーズを含むPCRチューブに添加した。これは、sstDNA 600,000コピーに相当する。
14. チューブを穏やかにボルテックス攪拌し、内容物を混合した。
15. MJサーモサイクラーのEPCRフォルダー中に保存されたプログラム80Annealを用いて、PCRサーモサイクラー中で、sstDNAを捕捉ビーズにアニーリングさせた。以下のプロトコールが用いられた。
・65℃で5分;
・0.1℃/秒で60℃に低下;
・60℃で1分間保持;
・0.1℃/秒で50℃に低下;
・50℃で1分間保持;
・0.1℃/秒で40℃に低下;
・40℃で1分間保持;
・0.1℃/秒で20℃に低下;および
・次の段階の用意ができるまで10℃で保持
17. 乳化まで、ビーズは氷バケツ中に保存した(実施例2)。
18. 捕捉ビーズには、1つのビーズあたり平均sstDNAが0.5から1コピー結合しており、乳化の準備ができた。
本実施例は、1マイクロリットルあたり約3,000のPCRマイクロリアクターを含む熱安定性の油中水エマルジョンを作製する方法を記述する。以下にエマルジョンの調製のためのプロトコールの概要を示す。
2. 溶液をピペットで数回出し入れして、ビーズを再懸濁した。
3. PCR-ビーズ混合液を室温で2分間インキュベートし、ビーズをPCR溶液で平衡化した。
4. 400μlのエマルジョン油をUV照射した2 mlマイクロ遠心チューブに添加した。
5. エマルジョン油のチューブに、「アンプリコンなし」の1/4"磁気攪拌バーを添加した。
・DNA-Off(液滴またはスプレー)で洗浄し;
・picopure水ですすぎ;
・キムワイプの端で乾燥させ;そして
・UVを5分間照射した。
7. チューブを充分にボルテックス攪拌し、ビーズを再懸濁した。これにより、凝集したビーズを最低限に抑えた。
8. P-200ピペットを用いて、次の液滴を添加する前に、各液滴が磁気攪拌バーのレベルに沈み、乳化するようにしながら、PCR-ビーズ混合物を2秒ごとに1滴の割合で1滴ずつ回転する油に添加した。溶液はマヨネーズと類似した粘性を有する、均一な乳白色の液体になった。
9. 全てのPCR-ビーズ混合物を添加したら、遠心チューブを数回はじき、表面にある油と乳状のエマルジョンを混合した。
10. さらに5分間攪拌を継続した。
11. 段階9および10を繰り返した。
12. より大きな攪拌バーを用いて、チューブから攪拌バーを引っ張り出すことにより、エマルジョンから攪拌バーを取り出した。
13. エマルジョン10μLを取り出し、顕微鏡スライド上に置いた。エマルジョンをカバースリップで覆い、50Xの倍率(接眼レンズ10X、対物レンズ5X)で観察した。「良い」エマルジョンは、油中にあるPCR溶液の分離した液滴(マイクロリアクター)中に単一のビーズが主に含まれているものである。
14. 以下のようにして、乳化安定剤を含む適当なエマルジョン油混合物が作製された。エマルジョン液の成分は、表4に示されている。
本実施例は、ビーズ-エマルジョン混合物における鋳型DNAの増幅を記述する。本発明のプロトコールに従うと、過程のDNA増幅段階は、3時間から4時間かかる。増幅の完了後、ビーズの単離過程を始める前に、エマルジョンは最高12時間、サーモサイクラー中に放置してもよい。PCRサーモサイクリングは、50から100μlの乳化反応液を個々のPCR反応チャンバー(すなわちPCRチューブ)に入れて実施された。PCRは以下のようにして行なわれた。
2. プレートを密封またはPCRチューブのフタを閉じ、96ウェルプレートアダプターを用いてまたは用いずに、容器をMJサーモサイクラー中に入れた。
3. PCRサーモサイクラーは、以下のプログラムで運転した:
・1サイクル(94℃で4分間)- ホットスタート;
・40サイクル(94℃で30秒間、58℃で30秒間、68℃で90秒間);
・25サイクル(94℃で30秒間、58℃で6分間);および
・14℃で保存
4. PCR反応完了後、エマルジョンの破壊およびビーズ回収に進むために、増幅産物を取り出した。
本実施例は、どのようにしてエマルジョンを破壊し、増幅鋳型を結合したビーズを回収するかを説明する。好ましくは、PCR後のエマルジョンは、完全な状態のままである。エマルジョンの下の方の層は、目視検査では乳白色の懸濁液のままである。溶液が透明な場合には、エマルジョンは水層と油層に部分的に分離した可能性があり、ビーズの多くが鋳型の混合物を有する可能性が高い。エマルジョンが1本または2本のチューブで破壊されている場合は、これらのサンプルは他のサンプルと混合しない。全てのチューブでエマルジョンが破壊されている場合は、本手順を継続しない。
2. 残ったエマルジョンは、各サンプルに50μlのSigma鉱油を添加して、各PCRチューブから回収された。単一のピペットチップを用いて、各チューブで数回出し入れして、残った材料を再懸濁した。
3. この材料を、乳化材料の大部分を入れた1.5 mlチューブに添加した。
4. サンプルを30秒間ボルテックスで攪拌した。
5. Eppendorfのテーブルトップマイクロ遠心機中で、13.2K rpmでサンプルを20分間遠心した。
6. エマルジョンは、大きな白色の界面を有する2つの層に分離した。上部の透明な油層をできるだけ多く除去した。混濁した材料は、チューブに残された。しばしば、白色の層が油層と水層を分離していた。ビーズはしばしばチューブの底に沈殿しているのが観察された。
7. ビーズの上の水層を取り出し、分析用(ゲル分析、Agilent2100、およびTaqman)に保存した。水層の上に白色の材料の界面が残る場合は、下の水層20μlが取り出された。これは、ピペットチップで界面材料を貫通し、その下から溶液を取り出すことによって行なった。
8. PTP製造および表面化学研究室のドラフト(PTP Fabrication and Surface Chemistry Room Fume Hood)内において、残りのエマルジョンに、1 mlのヘキサンが添加された。
9. サンプルを1分間攪拌し、1分間最高速度で遠心した。
10. PTP製造および表面化学研究室のドラフト内において、上部の油/ヘキサン層を除去し、有機廃液入れに入れた。
11. 1 mlの80%エタノール中の1Xアニーリング緩衝液を残りの水層、界面、およびビーズに添加した。
12. サンプルを1分間または白い物質が溶解するまでボルテックスで攪拌した。
13. サンプルを高速で1分間遠心した。チューブを180度回転させ、また1分間遠心した。ビーズの沈殿を乱さないようにして、上清を除去した。
14. 0.1% Tween20を含む1Xアニーリング緩衝液1 mlでビーズを洗浄し、この段階を繰り返した。
ピロリン酸ベースの配列決定反応にビーズが使用される場合には、PCR産物の2本目の鎖を除去し、ビーズに結合した一本鎖鋳型に配列決定プライマーをアニールさせる必要がある。本実施例は、そのためのプロトコールを記述する。
2. ビーズを1 mlの1 mM EDTAで洗浄した。チューブを段階1のようにして遠心し、水層を除去した。
3. 1 mlの0.125 M NaOHを添加し、サンプルを8分間インキュベートした。
4. サンプルを軽くボルテックスで攪拌し、マイクロ遠心機中にいれた。
5. 6分後、ビーズを段階1と同様に沈殿させ、できるだけ多くの溶液を除去した。
6. 8分間のNaOHインキュベーションの完了後、1 mlの1Xアニーリング緩衝液を添加した。
7. サンプルを軽くボルテックス攪拌し、ビーズを段階1と同様に沈殿させた。できるだけ多くの上清を除去し、さらに1 mlの1Xアニーリング緩衝液を添加した。
8. サンプルを軽くボルテックスで攪拌し、ビーズは段階1と同様に沈殿させ、800μlの1Xアニーリング緩衝液を除去した。
9. ビーズを0.2 ml PCRチューブに移した。
10. ビーズを移し、ビーズを乱さずにできるだけ多くのアニーリング緩衝液を除去した。
11. 100μlの1Xアニーリング緩衝液を添加した。
12. 4μlの100μM配列決定プライマーを添加した。アニーリングの直前に、サンプルをボルテックスで攪拌した。
13. アニーリングは「80Anneal」プログラムを用いて、MJサーモサイクラー中で行った。
14. 200μlの1Xアニーリング緩衝液でビーズを3回洗浄し、100μlの1Xアニーリング緩衝液中に再懸濁した。
15. Hausser 血球計算板でビーズを計数した。通常、300,000から500,000個のビーズが回収された(3,000〜5,000ビーズ/μL)。
16. ビーズは4℃で保存し、1週間は配列決定に使用できた。
以下の手順を用いてアンプリコンを含むビーズの濃縮を行なっても良い。濃縮は必要ではないが、この後のDNA配列決定のような分子生物学技術を、より効率良くするために使用できる。
以下の実験は、ビーズエマルジョンPCRの有効性を検定するために行われた。このプロトコールでは、平均直径25〜35μm(製造元の供給による)の600,000個のセファロースビーズを、ビーズ1つあたり3000万から5000万コピーの比率で、捕捉プライマーに共有結合した。捕捉プライマーが共有結合したビーズを、120万コピーの一本鎖アデノウイルスライブラリーと混合した。ライブラリー構築物は、ビーズ上の捕捉プライマーに相補的な配列を含んでいた。
実施例8:鋳型品質管理
既述のように、エマルジョンPCR反応の成功は、一本鎖鋳型種の質に関連することが分かった。したがって、エマルジョンPCRプロトコールを開始する前に、2つの異なる品質管理手段によって鋳型材料の質を評価した。まず、一本鎖鋳型の一部を2100 BioAnalyzer (Agilient)で分析した。サンプルに約200から500塩基のサイズの異質な断片集団が含まれていることを確認するために、RNA Pico Chipを使用した。第2に、Bio-Tek FL600プレート蛍光光度計でRiboGreen蛍光アッセイを用いて、ライブラリーを定量した。DNA濃度が5 ng/μlに満たないサンプルは、濃度が低過ぎて使用できないと考えられた。
1 mL N-ヒドロキシスクシンイミドエステル(NHS)-活性化セファロースHPアフィニティカラム(Amersham Biosciences、ピスカタウェイ、ニュージャージー州)に詰められたビーズをカラムから取り出した。30μmおよび25μmのポアフィルターメッシュセクション(Sefar America、デピュー、ニューヨーク州、米国)を連続して通過させることによって、30〜25μmサイズのビーズが選択された。第1のフィルターを通過したが、第2のフィルターに保持されたビーズを採集して、製品文献(Amersham Pharmaciaプロトコール#71700600AP)に記述されるようにして活性化した。増幅する鋳型
のセンスおよびアンチセンス鎖の5'末端に対応する2つの異なるアミン標識HEG(ヘキサエチレングリコール)長捕捉プライマーを得た。プライマーは、両端配列決定、すなわち、増幅産物の第1および第2の鎖の配列決定ができるように、増幅産物の両方の鎖を捕捉するように設計されている。捕捉プライマーは、20 mMリン酸緩衝液pH 8.0に溶解し、最終濃度を1 mMとした。各プライマー3μlをふるい分けた30〜25μmビーズに結合させた。その後、ビーズはビーズ保存緩衝液(50 mM Tris、0.02% Tweenおよび0.02%アジ化ナトリウム、pH 8)中で保存した。ビーズは血球計算板(Hausser Scientific、ホーシャム、ペンシルベニア州、米国)を用いて計数し、必要になるまで4℃で保存した。
他の全ての一本鎖分子の増幅技術と同様に、反応液に外来アンプリコンまたは他の実験からの残存アンプリコンが混入していると、配列決定に干渉する恐れがある。汚染の可能性を低下させるために、PCR反応混合物はPCRクリーンルームにあるUV処理の層流フード中で調製された。600,000ビーズのエマルジョンPCR反応1つあたり、1.5 mlチューブ中で以下の試薬が混合された:225μlの反応混合液(1XPlatinum HiFi緩衝液(Invitrogen))、1 mM dNTP、2.5 mM MgSO4 (Invitrogen)、0.1% BSA、0.01% Tween、0.003 U/μl熱安定性PPi-ase (NEB)、
および0.2 U/μl Platinum Hi-Fi Taqポリメラーゼ(Invitrogen)。25μlの反応液を取り出し、陰性対照として使用するために、200μlのPCRチューブ中に保存した。反応液および陰性対照の両方を、必要となるまで氷上で保存した。
配列決定のためのクローンDNA増幅の成功は、各ビーズに制御された数の鋳型種を送達することに関連している。以下に記述する実験では、典型的な標的鋳型濃度は、捕捉ビーズ1つあたり0.5鋳型コピーであると決定された。この濃度では、ポワソン分布によるとビーズの61%には鋳型が結合しておらず、30%には鋳型が1種結合し、9%には2つまたはそれ以上の鋳型種が結合している。過剰の鋳型種が送達されると、単一のビーズ上で混合した集団(2つまたはそれ以上の種)が結合し、そして増幅されることになり、意味のある配列データが得られなくなる。しかし、送達される種の数が少な過ぎると、鋳型を含むウェルの数が減り(ビーズ1つあたり1種)、配列決定のカバー範囲が減少する。したがって、一本鎖ライブラリー鋳型の濃度が重要であると考えられた。
乳化過程では、1μlあたり10,000の別々のPCRマイクロリアクターを含む熱安定性の油中水型エマルジョンが作製される。これは、単一分子、標的ライブラリーの個々の分子のクローン増幅のための基質となる。単一反応のための反応液およびDNA捕捉ビーズは、以下のようにして乳化された。UV処理した層流フードにおいて、200μlのPCR溶液(実施例10より)を600,000個のDNA捕捉ビーズ(実施例11より)を含むチューブに添加した。ビーズはピペッティングを繰り返して再懸濁した。その後、PCRビーズ混合液を室温で少なくとも2分間インキュベートし、ビーズとPCR溶液を平衡化した。同時に、450μlのエマルジョン油(軽油(Sigma)中、4.5% (w:w)Span 80、1% (w:w) Atlox 4912(Uniqema、デラウェア州))を、無菌の1/4インチ磁気攪拌バー(Fischer)を含む、フラットトップの2 mlの遠心チューブ(Dot Scientific)に分注した。その後、このチューブを特注のプラスチックチューブ保持ジグに入れ、これを450 RPMに設定したFisher Isotempデジタル攪拌ホットプレート(Fisher Scientific)の中央に入れた。
エマルジョンは、7〜8個の別々のPCRチューブに分注した。各チューブには、約75μlのエマルジョンが入れられた。チューブを密封し、上述の25μlの陰性対照と共に、MJサーモサイクラー中に入れた。以下のサイクルタイムを使用した:94℃で4分のインキュベーションを1サイクル(ホットスタート)、94℃で30秒間および68℃で150秒間のインキュベーションを30サイクル(増幅)、ならびに94℃で30秒間および68℃で360秒間のインキュベーションを40サイクル(ハイブリダイゼーションおよび伸長)。PCRプログラムの完了後に、チューブを取り出し、直ちにエマルジョンを破壊するか、反応液を破壊過程の開始まで最高16時間10℃で保存した。
増幅後、エマルジョンの破壊(油層と水層の分離)を検査した。破壊されていないエマルジョンは合わせて1.5 mlのマイクロ遠心チューブに入れ、時折見られる破壊されたエマルジョンは、廃棄した。エマルジョンサンプルの粘性が非常に高いので、各PCRチューブには、かなりの量が残った。チューブに残ったエマルジョンは、各PCRチューブに鉱油を75μl添加し、混合液をピペッティングして回収した。この混合液は乳化材料の大部分を含む1.5 mlのチューブに添加した。その後、1.5 mlチューブを30秒間ボルテックス攪拌した。その後チューブは、ベンチトップマイクロ遠心機中で、13.2K rpm(最高速度)で20分間遠心した。
ビーズの塊には、増幅され、固定されたDNA鎖を有するビーズ、および空または無効のビーズが含まれていた。上述のように、ビーズの61%は、増幅過程で鋳型DNAを持っていないと計算された。濃縮は、鋳型DNAを有するビーズを選択的に単離し、配列決定効率を最大化するために使用された。濃縮過程は以下に詳細に述べられている。
を2μl添加した。プライマーは、ビーズに固定された鋳型の3'末端の、結合した増幅および配列部位(各々20塩基の長さ)に相補的だった。溶液は中間の設定で2秒間ボルテックス攪拌することにより混合し、濃縮プライマーはMJサーモサイクラー中で制御された変性/アニーリングプログラムを用いて、固定されたDNA鎖にアニーリングした。プログラムでは、以下のサイクルタイムと温度が使用された:65℃で30秒間インキュベーション、58℃まで0.1/秒で低下、58℃で90秒間インキュベーション、および10℃で保持。
を3μl添加した。チューブを5秒間ボルテックス攪拌し、MJサーモサイクラーに設置し、以下の4段階アニーリングプログラムを行なった:65℃で5分間インキュベーション、50℃まで0.1℃/秒で低下、50℃で1分間インキュベーション、40℃まで0.1℃/秒で低下、40℃で1分間保持、15℃まで0.1℃/秒で低下、15℃で保持。
Claims (44)
- (a) 油中水型エマルジョンを形成して、複数の水性マイクロリアクターを作製する段階であって、マイクロリアクターの少なくとも1つが単一の核酸鋳型、核酸に結合できる単一のビーズ、および核酸増幅を行なうために必要な試薬を含む増幅反応溶液を含む段階;
(b) マイクロリアクター中で核酸を増幅して、核酸の増幅されたコピーを形成する段階;および
(c) 増幅されたコピーをマイクロリアクター中のビーズに結合させる段階
を含む、1つまたは複数の核酸の増幅方法。 - マイクロリアクターの大部分が単一の核酸を含む、請求項1記載の方法。
- 増幅反応溶液が、ポリメラーゼ連鎖反応の条件に適合する緩衝液中に懸濁されたヌクレオチド3リン酸、熱安定性ポリメラーゼ、および核酸プライマーを含むポリメラーゼ連鎖反応溶液である、請求項1記載の方法。
- ポリメラーゼ連鎖反応が、非対称ポリメラーゼ連鎖反応である、請求項3記載の方法。
- ポリメラーゼ連鎖反応が、対称ポリメラーゼ連鎖反応である、請求項3記載の方法。
- エマルジョンがさらに乳化安定剤を含む、請求項1記載の方法。
- 乳化安定剤が、Atlox 4912、Span 80、ならびにそれらの組み合わせおよび混合物からなる群より選択される、請求項6記載の方法。
- エマルジョンが熱安定性である、請求項1記載の方法。
- エマルジョンが95℃まで熱安定性である、請求項8記載の方法。
- 増幅が、転写ベースの増幅、cDNA末端の迅速な増幅、連続フロー式増幅(continuous flow amplification)、またはローリングサークル増幅(rolling circle amplification)からなる群より選択される方法によって実行される、請求項1記載の方法。
- エマルジョンが、核酸鋳型、ビーズ、および増幅反応溶液を油へ滴加することによって形成される、請求項1記載の方法。
- 少なくとも10,000の核酸を用いて行われる、請求項1記載の方法。
- 少なくとも50,000の核酸を用いて行われる、請求項1記載の方法。
- マイクロリアクターの平均サイズが直径50μm〜250μmである、請求項1記載の方法。
- 段階(c)の後に、各ビーズが核酸鋳型の10,000より多くの増幅コピーを捕捉している、請求項1記載の方法。
- 複数の核酸分子を含む単一の容器に入れられたライブラリーであって、各核酸分子が異なるビーズに別々に固定され、各ビーズが各核酸分子の1,000,000を超えるクローン増幅コピーを含む、ライブラリー。
- 核酸分子が、ゲノムDNA、cDNA、エピソームDNA、BAC DNA、およびYAC DNAからなる群より選択される、請求項16記載のライブラリー。
- ゲノムDNAが、動物、植物、ウイルス、細菌、および真菌のゲノムDNAからなる群より選択される、請求項16記載のライブラリー。
- ゲノムDNAがヒトゲノムDNAまたはヒトcDNAである、請求項18記載のライブラリー。
- ビーズの直径が2ミクロンから100ミクロンである、請求項16記載のライブラリー。
- ビーズがセファロースビーズである、請求項16記載のライブラリー。
- (a) 増幅される核酸鋳型を提供する段階;
(b) 核酸鋳型に結合できる、直径が約10μm〜約80μmの概ね球状のビーズを含む、固体支持体材料を提供する段階;
(c) 油中水型エマルジョンにおいて核酸増幅反応を実行するために必要な試薬を含む増幅反応溶液において、核酸鋳型とビーズを混合する段階;
(d) 核酸鋳型を増幅して核酸鋳型の増幅コピーを形成する段階;および
(e) ビーズに増幅したコピーを結合させる段階
を含む、核酸の増幅方法。 - (a) 核酸捕捉ビーズ;
(b) エマルジョン油;
(c) 1つまたは複数の乳化安定剤;
(d) 請求項1または請求項22記載の方法を実行するための説明書、
を含む、核酸鋳型の核酸増幅を実行するためのキット。 - 核酸が結合していないビーズから、核酸の増幅コピーに結合するビーズを濃縮する段階をさらに含み、濃縮段階がアフィニティ精製、電気泳動、および細胞ソーティングからなる群より選択される、請求項1または請求項22記載の方法。
- 濃縮段階が、核酸に結合する磁性ビーズを用いたアフィニティ精製によって実行される、請求項24記載の方法。
- 各ビーズに、少なくとも100,000コピーの各標的核酸分子が結合している、請求項1または22記載の方法。
- 各ビーズに、少なくとも1,000,000コピーの各標的核酸分子が結合している、請求項1または22記載の方法。
- 各ビーズに、少なくとも1〜20,000,000コピーの間の各標的核酸分子が結合している、請求項1または22記載の方法。
- ビーズがセファロースビーズである、請求項1または22記載の方法。
- 増幅コピーが、抗原/抗体、リガンド/受容体、およびポリヒスチジン/ニッケルからなる群より選択される結合対によってビーズに結合する、請求項1または22記載の方法。
- 結合対がアビジン/ビオチンである、請求項30記載の方法。
- 鋳型を有するビーズと磁性ビーズを分離する段階;および
磁場を用いて磁性ビーズを除去する段階、
をさらに含む、請求項25記載の方法。 - 分離が、45℃を超える温度でのインキュベーションまたは鋳型を有するビーズと磁性ビーズを塩基性pHの溶液中でインキュベートすることにより達成される、請求項32記載の方法。
- (a) 長さが50〜800 bpの複数の核酸鋳型および核酸鋳型に結合できるビーズを提供する段階;
(b) 核酸鋳型の増幅に必要な試薬を含む生物学的反応溶液中で、核酸鋳型とビーズを混合する段階;
(c) エマルジョンを形成して、核酸鋳型、ビーズ、および生物学的反応溶液を含む複数のマイクロリアクターを作製する段階であって、少なくとも1つのマイクロリアクターが生物学的反応溶液中に封入された単一の核酸鋳型および単一のビーズを含み、マイクロリアクターが同一の容器中に入れられている、段階
を含む、核酸のクローン集団の生成方法。 - 核酸の転写および翻訳を行い、少なくとも10,000コピーの発現産物を産出するための段階をさらに含む、請求項34記載の方法。
- 抗原/抗体、リガンド/受容体、6Xhis/ニッケル-ニトリロ3酢酸、およびFLAG タグ/FLAG抗体結合対からなる群より選択される結合対によって、発現産物がビーズに結合している、請求項35記載の方法。
- 蛋白質のクローン集団を生成する、請求項35記載の方法。
- 蛋白質が抗体、抗体断片、および操作された抗体からなる群より選択される、請求項37記載の方法。
- 直径が50μm〜200μmであって生物学的反応溶液を含む多数の熱安定性マイクロリアクターを含むエマルジョン。
- 生物学的反応溶液が、ポリメラーゼ連鎖反応増幅を実行するための試薬を含む、請求項39記載のエマルジョン。
- 生物学的反応溶液が、転写と翻訳のカップリング反応を実行するための試薬を含む、請求項39記載のエマルジョン。
- 複数のマイクロリアクターが核酸鋳型を含む、請求項40または41記載のエマルジョン。
- 複数のマイクロリアクターが、1つまたはそれより少ない核酸鋳型を含む、請求項42記載のエマルジョン。
- 複数のマイクロリアクターが、核酸鋳型に結合する1つまたはそれより少ないビーズを含む、請求項43記載のエマルジョン。
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