JP2007527214A - 遺伝的変異の検出および列挙のための方法ならびに組成物 - Google Patents
遺伝的変異の検出および列挙のための方法ならびに組成物 Download PDFInfo
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Abstract
Description
本発明は遺伝分析の分野に関する。特に、個々の遺伝子または転写産物における変異を分析すること、および変異体を分離することのための方法ならびに組成物に関する。
DNA配列変異の研究は多くの研究領域にとって不可欠である。生殖系列変異の研究は、正常および異常な生理的状態における遺伝の役割を評価するために不可欠である(1)。体細胞性に発生した他の変異は腫瘍症の原因である(2)。それゆえに、尿、痰および大便におけるそのような突然変異の同定は、前駆症状性癌の検出のために用いられうる(3-5)。同様に、リンパ節、血液または骨髄における体細胞性突然変異の検出は、病期、予後、および様々な治療の適合性についてのデータを提供することができる(5)。非腫瘍性細胞における体細胞性突然変異もまた生じ、人間が加齢するまたは環境危険に曝されるために、蓄積するようにみえる(6)。そのような突然変異は、組織における細胞のほんの小部分にのみ生じ、それによりそれらの分析を困難にしている。
本発明の第一態様において、組成物が提供される。組成物は、複数のビーズを含む。複数のビーズのそれぞれは、複数の結合したポリヌクレオチドを含む。組成物におけるポリヌクレオチドは不均一である;しかしながら、そのビーズの少なくとも1%において、複数の結合したポリヌクレオチドは均一である。
本発明者らは、何百万個という分子を評価する能力をもち、一般的に、遺伝的変異の研究に適用されうる、BEAMingと呼ばれるデジタルテクノロジーを本明細書に記載する。テクノロジーは、単一DNA分子の、最初のDNA分子の配列の何千個というコピーをそれぞれに含む単一ビーズへの変換を含む。集団における変異体DNA分子の数は、その後、例えば、ビーズを蛍光ビーズで染色し、フローサイトメトリーを用いてそれらを数えることにより評価されうる。特定の変異体を表すビーズは、任意で、フローソーティングを通して回収され、その後の確認および実験に用いられうる。
実施例1--材料および方法
段階1 - オリゴヌクレオチドのビーズへの結合。直径1.05+/-0.1 umのストレプトアビジンに共有結合性に結合した超常磁性ビーズをDynal Biotech, Inc. (650.01, Lake Success, NY)から購入した。ビーズを1x PCR緩衝液(53286, Invitrogen, Carlsbad, CA)で1回、洗浄し、その後、Bind and Wash Buffer(BWB)(5 mM トリス-HCl、0.5 mM EDTA、1.0 M NaCl、pH 7.5)に懸濁した。ビーズは、10 uM オリゴヌクレオチドの存在下、室温で30 min、BWBにおいてインキュベートした(図8)。これらのオリゴヌクレオチドは、6炭素リンカーにより分離されたビオチン基をもつ5'末端における2重のビオチン基(IDT, Coralville, IA)で修飾された。結合後、ビーズを1x PCR緩衝液で3回、洗浄し、結合していないオリゴヌクレオチドを完全に除去した。
段階1 - オリゴヌクレオチドのビーズへの結合。本発明者らは、ストレプトアビジン-ビーズを、ビオチン化オリゴヌクレオチドをそれらに結合することの簡単さという理由で用いた。ただ単一の5'ビオチン基をもつオリゴヌクレオチドは、温度サイクリング中にビーズから解離することが見出されたが、それらの5'末端に二重ビオチン基(6炭素リンカーにより分離された)で標識されたオリゴヌクレオチドはサイクリングに対して安定していた。6-FAMおよびビオチンで二重に標識されたオリゴヌクレオチドの蛍光透視測定により測定される場合、約105個のオリゴヌクレオチド分子が各ビーズに結合していた。本発明者らは、短いオリゴヌクレオチド(20塩基)がより長いもの(41 bp)ほどプライミングについて十分に働かないことを見出したが、おそらく、ビーズ表面における立体障害のためと思われる。対応する反応基で修飾されたビーズへ共有結合性に結合したアミノ-、スルフヒドリル-、またはカルボキシ-修飾されたオリゴヌクレオチドもまた、BEAMingのためのビーズ結合プライマーとして機能することができる可能性が高い。
パイロット実験は、材料および方法に記載された水-油混合物を単に撹拌することが、ビーズのサイズと適合するサイズの非常に安定したマイクロエマルジョンを生成することを実証した。図2に示された実験において、水性コンパートメントは、青色色素、および5'末端でビオチン化され、かつ3'末端でフルオレセインで標識されたオリゴヌクレオチドに結合することにより標識された1ミクロンの磁気ビーズを含んだ。それらの形成直後のエマルジョンの外観は図2に示されている。予想どおり、この外観は、PCR中の温度サイクリング後、変化しなかった(15)。前のセクションで提供された図から予想されるとおり、たいていの水性コンパートメントはビーズを含まなかった。ビーズを含むそれらのコンパートメントは、一般的に、1個のみを含んだが、ポアソン分布および一様ではない水性コンパートメントサイズから予想されるとおり、画分はより多くを含んだ。両方の対立遺伝子を示すPCR産物を含む「ヘテロ接合性」ビーズは、2個またはそれ以上のDNA鋳型分子が単一の水性コンパートメント内に含まれる場合に生じる。そのようなヘテロ接合体は、場合によっては分析の精度を損なうことがある(考察参照)。
図3は、ヒトDNA試料で得られた典型的結果を示す。この実験で用いられたMID42マーカーは、Weberおよび同僚により構築された二対立遺伝子の短い挿入/欠失多型のコレクションから選択された(17)。これらの対立遺伝子は、各座位における2つの対立遺伝子が約4塩基だけ異なるため、ハイブリダイゼーションプローブで識別することは特に簡単である。長い方(L)の対立遺伝子についてのプローブは、フルオレセイン(緑色)で標識され、短い方(S)の対立遺伝子についてのプローブは、R-フィコエリトリン(赤色)で標識された。
図3に示された結果は、ヒトゲノムDNA試料から作製されたPCR産物を用いて生じた。S対立遺伝子を表すビーズに対するL対立遺伝子を表すビーズの比率はこの実験において1.0であったため、最初のPCRはどちらの対立遺伝子も優先的には増幅しなかったことは明らかであった。ゲノムDNAよりむしろPCR産物の使用は、多数の対立遺伝子が少量の出発DNAでさえからも増幅されるのを可能にする。一般的に、サイズ200 bpのPCR産物の10〜100ピコグラムが、約1%から10%までの標識ビーズにおいて、BEAMing、プライマーのPCR仲介型伸長を生じるために最適であることが見出された。
まれな変異の分析は、高いシグナル対ノイズ比を保持しながら、独立して分析されうる多数の分子のために、BEAMingによる分析に理想的に適している。図5Aは、MID42のL対立遺伝子の1%、2%、3%および4%を表す鋳型からの代表的データを示す。0.99の相関係数をもつこれらの測定の線形性は、そのような適用のためのこのアプローチの有用性を実証している。本発明者らはまた、この分析をKRASの検出に適用し、野生型分子の集団へスパイクした場合、0.1%突然変異体を容易に観察することができた(データ示さず)。
100 bp産物を実施例1、段階1〜4に記載されているようにビーズ上で増幅した。2つのFAM標識オリゴヌクレオチド(50塩基長および20塩基長)を、ビーズ上の100 bp産物へアニールした。その後、ビーズを、卵形の配置でアクリルアミドゲルに包埋した(通常のトリス-ホウ酸-EDTA電気泳動緩衝液を用いて)。電場(250 V)は、変性条件下で3分間加えられた。標識されたオリゴヌクレオチドは、ビーズから離れて移動し、それらのサイズに関連した距離を移動した。図7を参照。ビーズの卵形の保持により証明されているように、ほとんど拡散はなかった。
サンガー型(ジデオキシヌクレオチド)シーケンシングは、実施例1に記載されているように、ビーズ上で増幅されたオリゴヌクレオチドを鋳型として用いて行われた。個々のビーズを、ジデオキシヌクレオチド阻害剤の存在下でプライマー伸長条件にかける。その後、ビーズを、変性条件下で電気泳動にかけ、ジデオキシヌクレオチド終結したプライマー伸長オリゴヌクレオチドを長さに基づいて分離する。配列は、プライマー伸長オリゴヌクレオチドの長さに基づいて編集される。
Claims (63)
- 複数のビーズを含む組成物であって、該複数のビーズのそれぞれが複数の結合したポリヌクレオチドを含み、組成物におけるポリヌクレオチドが不均一であり、かつ該ビーズの少なくとも1%における複数の結合したポリヌクレオチドが均一である、組成物。
- ビーズの少なくとも5%における複数の結合したポリヌクレオチドが均一である、請求項1記載の組成物。
- ビーズの少なくとも10%における複数の結合したポリヌクレオチドが均一である、請求項1記載の組成物。
- ビーズの少なくとも50%における複数の結合したポリヌクレオチドが均一である、請求項1記載の組成物。
- 複数の結合したポリヌクレオチドが100個より多い、請求項1記載の組成物。
- 液体である、請求項1記載の組成物。
- アガロースを含む、請求項6記載の組成物。
- 組成物におけるポリヌクレオチドが一塩基多型(SNP)により異なる、請求項1記載の組成物。
- 組成物におけるポリヌクレオチドが突然変異の存在または非存在の点で異なる、請求項1記載の組成物。
- 組成物におけるポリヌクレオチドが挿入の存在または非存在の点で異なる、請求項1記載の組成物。
- 組成物におけるポリヌクレオチドが多型の存在または非存在により異なる、請求項1記載の組成物。
- ビーズが磁性である、請求項1記載の組成物。
- ポリヌクレオチドの少なくとも1種が蛍光色素で標識されている、請求項1記載の組成物。
- 標識されたオリゴヌクレオチドを介して標識化が行われている、請求項13記載の組成物。
- 1つまたは複数の標識された抗体を介して標識化が行われている、請求項13記載の組成物。
- 結合したポリヌクレオチドが試験試料における鋳型の増幅により作製され、複数の結合したポリヌクレオチドが均一であるビーズが、ポリヌクレオチドの少なくとも第一種および第二種を含み、ポリヌクレオチドの第一種を含むビーズおよびポリヌクレオチドの第二種を含むビーズが、ポリヌクレオチドの第一種および第二種が試験試料において存在しているのと同じ比率で組成物に存在している、請求項1記載の組成物。
- 水性コンパートメントを形成する複数のマイクロエマルジョンを含む液体組成物であって、該水性コンパートメントの少なくとも一部は以下のものを含み:
ビーズ;
ポリヌクレオチド鋳型;および
該鋳型を増幅するためのオリゴヌクレオチドプライマー;
オリゴヌクレオチドプライマーの少なくとも一部はビーズに結合している、液体組成物。 - フォワードおよびリバースオリゴヌクレオチドプライマーを含む、請求項17記載の液体組成物。
- 水性コンパートメントが0.5ミクロン〜50ミクロンの平均直径を有する、請求項17記載の液体組成物。
- 水性コンパートメントの10,000個のうち少なくとも1個がビーズを含む、請求項17記載の液体組成物。
- 水性コンパートメントの1/100から1までがビーズを含む、請求項17記載の液体組成物。
- 水性コンパートメントの1/50から1までがポリヌクレオチド鋳型分子を含む、請求項17記載の液体組成物。
- ビーズが磁性である、請求項17記載の液体組成物。
- 水性コンパートメントあたりの鋳型分子の平均数が1未満である、請求項17記載の液体組成物。
- DNAポリメラーゼおよびデオキシリボヌクレオチドをさらに含む、請求項17記載の液体組成物。
- 水性コンパートメントの平均直径が1ミクロン以上10ミクロン以下である、請求項17記載の液体組成物。
- 水性コンパートメントの平均直径が11ミクロン以上100ミクロン以下である、請求項17記載の液体組成物。
- 水性コンパートメントの平均直径が約5ミクロンである、請求項17記載の液体組成物。
- 各オリゴヌクレオチドプライマーが少なくとも12ヌクレオチド長である、請求項17記載の液体組成物。
- 各オリゴヌクレオチドプライマーが25ヌクレオチドから55ヌクレオチドである、請求項17記載の液体組成物。
- オリゴヌクレオチドプライマーのビーズへの結合が共有結合性である、請求項17記載の液体組成物。
- ビオチン-ストレプトアビジン結合ペアを介してオリゴヌクレオチドプライマーがビーズへ結合している、請求項17記載の液体組成物。
- ビーズに結合しているフォワードまたはリバースオリゴヌクレオチドプライマーが少なくとも2つのビオチン部分を含む、請求項32記載の液体組成物。
- 水性コンパートメントがアガロースを含む、請求項17記載の液体組成物。
- 以下の段階を含む、ヌクレオチド配列変異を分析するための方法:
分析物DNA分子の1つまたは複数の種を含むマイクロエマルジョンを形成する段階;
試薬ビーズの存在下においてマイクロエマルジョンにおける分析物DNA分子を増幅する段階であって、試薬ビーズが、分析物DNA分子を増幅するためのプライマーの複数の分子に結合しており、分析物DNA分子の1つの種の複数のコピーに結合している生成物ビーズが形成される、段階;
生成物ビーズを、生成物ビーズに結合していない分析物DNA分子から分離する段階;
生成物ビーズに結合している分析物DNA分子の1つの種の配列特徴を決定する段階。 - 分析物DNA分子の第一種の複数のコピーに結合している生成物ビーズを、分析物DNA分子の第二種の複数のコピーに結合している生成物ビーズから単離する段階をさらに含む、請求項35記載の方法。
- 単離段階が蛍光活性化セルソーティングを用いて行われる、請求項36記載の方法。
- 分析物DNA分子の第一種を生成物ビーズから回収する段階をさらに含む、請求項36記載の方法。
- 単離された生成物ビーズ由来の分析物DNA分子の第一種を増幅する段階をさらに含む、請求項36記載の方法。
- 分析物DNA分子の第一種の配列を決定する段階をさらに含む、請求項38記載の方法。
- 増幅段階が、マイクロエマルジョンに存在する試薬ビーズの10%未満を生成物ビーズへ変換する、請求項35記載の方法。
- 分離段階の前に、マイクロエマルジョンが1つまたは複数の界面活性剤の添加により破壊される、請求項35記載の方法。
- 決定段階が、異なって標識されているオリゴヌクレオチドプローブへのハイブリダイゼーションにより行われる、請求項35記載の方法。
- 1つまたは複数の配列特徴を含む生成物ビーズの相対的または絶対的量が測定される、請求項35記載の方法。
- 相対的または絶対的量がフローサイトメトリーを用いて測定される、請求項44記載の方法。
- 増幅段階が、試薬ビーズに結合していないプライマーの追加のコピーを用いる、請求項35記載の方法。
- 分析物DNA分子がゲノムDNAである、請求項35記載の方法。
- 分析物DNA分子がcDNAである、請求項35記載の方法。
- 分析物DNA分子がゲノムDNAから作製されたPCR産物である、請求項35記載の方法。
- 分析物DNA分子がcDNAから作製されたPCR産物である、請求項35記載の方法。
- 分析物DNA分子が単一個体由来である、請求項35記載の方法。
- 分析物DNA分子が個体の集団由来である、請求項35記載の方法。
- 試薬ビーズが磁性である、請求項35記載の方法。
- 配列特徴を決定する段階が、1つまたは複数の標識デオキシリボヌクレオチドでのプライマーの伸長により行われる、請求項35記載の方法。
- 以下のものを含む、固体支持体に結合しているポリヌクレオチドへのハイブリダイゼーションにおける使用のためのプローブ:
5'末端または3'末端の1つにホトルミネセンス色素を有するステム-ループ構造をもつオリゴヌクレオチドであって、反対側の5'末端または3'末端に消光剤を含まない、オリゴヌクレオチド。 - 突然変異体選択遺伝子配列へよりも良く野生型選択遺伝子配列へハイブリダイズする、請求項55記載のプローブ。
- 野生型遺伝子配列へよりも良く突然変異体遺伝子配列へハイブリダイズする、請求項55記載のプローブ。
- 以下のものを含む、分子プローブのペア:
5'末端または3'末端の1つに第一ホトルミネセンス色素を有するステム-ループ構造をもつ第一オリゴヌクレオチドであって、反対側の5'末端または3'末端に消光剤を含まず、突然変異体選択遺伝子配列へよりも良く野生型選択遺伝子配列へハイブリダイズする、第一オリゴヌクレオチド;および
5'末端または3'末端の1つに第二ホトルミネセンス色素を有するステム-ループ構造をもつ第二オリゴヌクレオチドであって、反対側の5'末端または3'末端に消光剤を含まず、野生型選択遺伝子配列へよりも良く突然変異体選択遺伝子配列へハイブリダイズする、第二オリゴヌクレオチド;
ここで、第一および第二ホトルミネセンス色素は異なる。 - 以下の段階を含む、ヌクレオチド配列変異体を単離するための方法:
分析物DNA分子の1つまたは複数の種を含むマイクロエマルジョンを形成する段階;
試薬ビーズの存在下においてマイクロエマルジョンにおける分析物DNA分子を増幅する段階であって、試薬ビーズが、分析物DNA分子を増幅するためのプライマーの複数の分子に結合しており、分析物DNA分子の1つの種の複数のコピーに結合している生成物ビーズが形成される、段階;
生成物ビーズを、生成物ビーズに結合していない分析物DNA分子から分離する段階;
分析物DNA分子の第一種の複数のコピーに結合している生成物ビーズを、分析物DNA分子の第二種の複数のコピーに結合している生成物ビーズから単離する段階。 - 単離段階が蛍光活性化セルソーティングを用いて行われる、請求項59記載の方法。
- 分析物DNA分子の第一種を生成物ビーズから回収する段階をさらに含む、請求項59記載の方法。
- 単離された生成物ビーズ由来の分析物DNA分子の第一種を増幅する段階をさらに含む、請求項59記載の方法。
- 分析物DNA分子の第一種の配列を決定する段階をさらに含む、請求項59記載の方法。
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CN1950519A (zh) * | 2004-02-27 | 2007-04-18 | 哈佛大学的校长及成员们 | 聚合酶群落荧光原位测序珠子 |
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JP2017012080A (ja) * | 2015-06-30 | 2017-01-19 | シスメックス株式会社 | 遺伝子検査用検体処理装置 |
JP2017143783A (ja) * | 2016-02-17 | 2017-08-24 | 国立大学法人 筑波大学 | 並列反応用懸濁液、並列反応方法、スクリーニング方法および検査方法 |
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US10604797B2 (en) | 2020-03-31 |
CA2531105C (en) | 2015-03-17 |
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EP1641809A2 (en) | 2006-04-05 |
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EP2532745A2 (en) | 2012-12-12 |
EP2918595A2 (en) | 2015-09-16 |
WO2005010145A2 (en) | 2005-02-03 |
JP5183063B2 (ja) | 2013-04-17 |
HK1179304A1 (en) | 2013-09-27 |
EP2532745B1 (en) | 2015-09-09 |
US20160319341A1 (en) | 2016-11-03 |
US9328343B2 (en) | 2016-05-03 |
US20120183967A1 (en) | 2012-07-19 |
WO2005010145A3 (en) | 2005-08-11 |
EP2532745A3 (en) | 2014-03-12 |
CA2531105A1 (en) | 2005-02-03 |
EP1641809B2 (en) | 2018-10-03 |
US8048627B2 (en) | 2011-11-01 |
US20090286687A1 (en) | 2009-11-19 |
EP2918595A3 (en) | 2015-10-21 |
EP1641809A4 (en) | 2010-05-19 |
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