JP2018516074A - クラスターにおける表面プライマーの高度な利用 - Google Patents
クラスターにおける表面プライマーの高度な利用 Download PDFInfo
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- JP2018516074A JP2018516074A JP2017558661A JP2017558661A JP2018516074A JP 2018516074 A JP2018516074 A JP 2018516074A JP 2017558661 A JP2017558661 A JP 2017558661A JP 2017558661 A JP2017558661 A JP 2017558661A JP 2018516074 A JP2018516074 A JP 2018516074A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
Description
シークエンシング手法には、第1リードの対向鎖上に第2シークエンシングリードを含むペアエンドシークエンシングを含む場合があり、例えば、本明細書において参照によりそれぞれ組み込む米国特許第7,754,429号明細書及び同第8,017,335号明細書に記載されている。一般的な実施形態において、ペアエンド法は、2表面結合プライマーにより、シークエンシングした鎖のコピーを生成できる点で有利である。この、相補鎖を再生するプロセスは、多くの場合、ペアエンドターンと呼ばれる。しかしながら、上述した各方法においては、固体支持体の表面から2種のプライマーのうちの1つを開裂するものであり、従来の手法を用いるペアエンドアプローチは不可能な場合がある。
本明細書に示した方法及び組成物において、ポリヌクレオチドは固体支持体に固定化されている。いくつかの実施形態において、ポリヌクレオチドは共有結合的に支持体に固定化されている。分子(例えば、核酸)の支持体への固定化というとき、本明細書における用語「固定化(immobilized)」及び「付着(attached)」は、それぞれ、区別なく用いられており、明示的又は文脈により特に指示しない限り、直接結合、間接結合、共有結合、又は非共有結合を包含することを意図している。本発明の特定の実施形態において、共有結合が好ましい場合があるが、一般に、支持体を使用しようとする条件下、例えば、核酸増幅及び/又はシークエンシングを必要とする用途において、分子(例えば、核酸)の支持体への固定化又は結合を維持することができればよい。
例えば、いくつかの実施形態において、米国特許第7,985,565号明細書及び同7,115,400号明細書に例示されるクラスター増幅法を用いて固定化DNA断片を増幅する。各文献の内容は、本明細書においてその全体を参照により組み込む。この組み込んだ米国特許第7,985,565号明細書及び同7,115,400号明細書の各文献には、クラスター又は「コロニー」を含むアレイを形成するため、増幅産物を固体支持体上で固定可能な固相核酸増幅方法が記載されている。そのようなアレイ上の各クラスター又はコロニーは、それぞれ、複数の同一の固定化ポリヌクレオチド鎖及び複数の同一の固定化相補ポリヌクレオチド鎖から形成する。このように形成したアレイは、本明細書において、広く「クラスター化アレイ」と称する。米国特許第7,985,565号明細書及び同7,115,400号明細書に記載されるような固相増幅反応の産物は、固定化ポリヌクレオチド鎖と固定化相補鎖の各対をアニーリングすることにより形成したいわゆる「ブリッジ化」された構造である。固定化ポリヌクレオチド鎖及び固定化相補鎖はいずれも、5’末端において固体支持体上に、好ましくは、共有結合を介して固定されている。クラスター増幅法は、固定化核酸鋳型を用いて固定化アンプリコンを製造する方法の例示である。他の好適な方法を用いて、本明細書に記載の方法により生成した固定化DNA断片から固定化アンプリコンを製造することもできる。例えば、各増幅プライマー対のプライマーの一方又は両方が固定化されているか否かに関わらず、固相PCRを介して1つ以上のクラスター又はコロニーを形成することができる。
本明細書に記載の各方法は、種々の核酸シークエンシング方法と共に用いることができる。特に適用可能な技術としては、アレイ中の固定位置に核酸を付着して、各核酸の相対的な位置が変わらないようにし、そのアレイを繰り返し撮像するものである。異なるカラーチャネルにおいて画像が取得される実施形態においては、例えば、一のヌクレオチド系種を他から識別するのに使用する異なる標識と一致することが特に適用可能である。いくつかの実施形態において、標的核酸のヌクレオチド配列を判定するプロセスは、自動化することができる。好ましい実施形態としては、1塩基合成((Sequencing by Synthesis(SBS)法がある。
シークエンシングの第1のサイクルを用いた増幅方法の比較分析
本実施例は、溶液中にプライマーを添加した場合と添加しない場合とにおける、標準ExAmp増幅と、その後のプライマー開裂イベント及び部分的変性条件下での増幅を含む他の方法との比較を示すものである。
シークエンシングの第1のサイクルを用いた増幅方法の比較分析
実施例1において説明した分析に続いて、フローセルをその後作製して、シークエンシングプライマーをハイブリダイズし、イルミナ製組込ミックスIMXを用いて5分間55℃で洗浄して、第1のシークエンシング組込を行った。組込ミックスは、ポリメラーゼ、及び3’−ブロック化dNTPの標識化ミックスを含む。イルミナ製洗浄バッファPR2で洗浄後、トリス0.1M/アスコルビン酸ナトリウム0.1Mのスキャンミックスをフローセルに流し、第1のサイクル画像を、図4に示すように、蛍光顕微鏡で撮影した。さらに、各レーンにおけるクラスターのCy3染色及びCy5染色を定量化することにより撮像分析を行った。図4の下部パネル画に示すように、定量化により、横方向ブースト(レーン2)だけでも、コントロールと比べて少なくとも2倍明瞭なクラスターを生成することが分かる。溶液プライマー(レーン3)を用いた横方向ブーストにより、コントロールと比べて6倍以上明瞭なクラスターを生じる。
Claims (11)
- 核酸シークエンシング反応のための固定化鋳型を作成する方法であって、
(a)固体支持体を設けるステップであって、該固体支持体はその上に固定化した複数の順方向増幅プライマー及び逆方向増幅プライマーを有し、前記複数の増幅プライマーのサブセットは開裂部位を含む、固体支持体上を設けるステップ、
(b)支持体上のプライマーのサブセットを用いて鋳型を増幅し、複数の二本鎖核酸分子を生成するステップであって、各二本鎖核酸分子の両鎖はその5’末端にて固体支持体に結合される、生成するステップ、
(c)開裂部位にてプライマーのサブセットを開裂するステップ、及び
(d)部分変性条件下に開裂鎖を置いて、増幅産物の非固定化鎖のハイブリダイゼーションを促進した後、固定化増幅プライマーを伸長して、増幅産物の非固定化鎖のコピーを生成するステップ、
を含む、方法。 - 部分変性条件は、リコンビナーゼ/ポリメラーゼ増幅反応の成分を1つ以上添加してストランド侵入を促進させることを含む、請求項1に記載の方法。
- 部分変性条件は、鋳型ウォーキングに適した条件下に鋳型を置くことを含む、請求項1に記載の方法。
- ステップ(d)は、溶液中にプライマーを加えて、固定化増幅産物の非固定化端部まで該プライマーのハイブリダイゼーションを促進することを含む、請求項1に記載の方法。
- プライマーのサブセットは順方向増幅プライマーを含み、溶液中のプライマーは順方向増幅プライマーを含む、請求項4に記載の方法。
- プライマーのサブセットは逆方向増幅プライマーを含み、溶液中のプライマーは逆方向増幅プライマーを含む、請求項4に記載の方法。
- 標的核酸をシークエンシングするステップをさらに含む、請求項1〜6のいずれかに記載の方法。
- 標的核酸をシークエンシングするステップは、
1つ以上のシークエンシングプライマーを第1の固定化鋳型又は第2の非固定化鎖にハイブリダイズするステップ、
1つ以上の標識ヌクレオチドを新生鎖に取り込むことにより、シークエンシングプライマーを伸長するステップ、及び
標識ヌクレオチドを検出することにより、標的核酸についての配列情報を得るステップ、を含む、請求項7に記載の方法。 - 固体支持体は平坦である、請求項1〜8のいずれかに記載の方法。
- 固体支持体はマイクロウェルを備える、請求項1〜9のいずれかに記載の方法。
- 標的核酸は、少なくとも10、20、50、100、200、又は少なくとも500ヌクレオチド分の長さを有する、請求項1〜10のいずれかに記載の方法。
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