JP6724287B2 - 核酸の配列決定方法及びシステム - Google Patents
核酸の配列決定方法及びシステム Download PDFInfo
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- JP6724287B2 JP6724287B2 JP2018523734A JP2018523734A JP6724287B2 JP 6724287 B2 JP6724287 B2 JP 6724287B2 JP 2018523734 A JP2018523734 A JP 2018523734A JP 2018523734 A JP2018523734 A JP 2018523734A JP 6724287 B2 JP6724287 B2 JP 6724287B2
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Description
本明細書では、鋳型核酸分子の配列決定方法を提供する。本方法は、一般的に、非標識ヌクレオチドの組込みの前に検査工程を含む。より具体的には、検査工程は、プライマーでプライミングした鋳型核酸分子を提供し、プライミングした鋳型核酸分子を、ポリメラーゼ及び少なくとも1つの非標識ヌクレオチド分子を含む第一の反応混合物に接触させ、非標識ヌクレオチド分子の存在下で、プライミングした鋳型核酸分子への核酸分子の化学的組込みを伴わずに、ポリメラーゼとプライミングした鋳型核酸との相互作用をモニタリングし、ならびに、非標識ヌクレオチド分子の存在下で、モニタリングしたポリメラーゼとプライミングした鋳型核酸分子との相互作用に基づいて、鋳型核酸中の後続塩基を同定することを含む。
ハイスループットで費用対効果の高い核酸の配列決定は、研究と個別化医療の新しい時代を導く可能性を秘めている。いくつかの市販の配列決定プラットフォームが利用可能であり、それらはゲノム全体の規模で配列決定を行うが、遺伝子解析の大量消費市場においてまだまだ高価である。配列決定コストを削減することにより、種間及び個体間の遺伝的変異を詳細に解析し、個別化医療の基礎を提供し、ならびに遺伝子型と表現型の間の関連性を特定することが可能になる。試薬と人件費の削減に加えて、配列決定技術の目標にはスループットの拡大と精度の向上が含まれる。
方法及び材料。ポリメラーゼ緩衝液:20mMトリス、pH8、300mM NaCl、5mM DTT、100μM dNTP、150nM Klenow、0.01% BSA、0.02% tween−20、10mM MgCl2。検査緩衝液:20mMトリス、pH8、300mM NaCl、5mM DTT、100μM dNTP、150nM Klenow、0.01% BSA、0.02% tween−20。組込み緩衝液:20mMトリス、pH8、300mM NaCl、5mM DTT、0.01% BSA、0.02% tween−20,10mM MgCl2。洗浄緩衝液:20mMトリス、pH8、300mM NaCl、5mM DTT、0.01% BSA、0.02% tween−20。
結合反応速度を用いて、検査工程中、すなわちポリメラーゼ、DNA鋳型、及びヌクレオチドの間の三元複合体の形成中に正しい塩基またはヌクレオチドを決定することができる。これを図2に示す。Bst酵素は、正しい塩基が存在する場合には二峰性の結合曲線を示し、誤った塩基が存在する場合には指数関数的な結合挙動を示す(図2)。したがって、配列決定中に正しい塩基またはヌクレオチドの識別及び検出が可能になる。
本実施例では、検査工程の間に高濃度の塩を用いて配列決定反応を行い、次いで組込み工程を行った。
FORTEBIO(登録商標)Octet instrument(Red 384またはqk)(メンローパーク、カリフォルニア州)は、バイオレイヤー干渉法を使用して、光ファイバーチップの表面で結合反応を測定する。本実施例では、チップをストレプトアビジン(SA)で官能化し、鋳型の3′末端付近の配列に相補的なプライマーとハイブリダイズした5′ビオチン標識DNA鋳型に結合できるようにした。
会合工程中及び解離工程中の両方において検査を共に使用して、配列の忠実度を改善することができる。したがって、解離反応速度を評価し、その評価を用いて塩基の同一性を決定し、配列の忠実度を高めることができるかどうかを判定した。
ホモポリマー領域における複数回の付加の可能性を最小限に抑えるために、組込み前に洗浄工程を実施することができる。カルシウム及びストロンチウムなどの金属陽イオンは、マグネシウムのようなポリメラーゼ複合体を安定化することができるが、ヌクレオチドの組込みをもたらすホスホジエステル結合の触媒作用をサポートすることができない。本実験では、洗浄緩衝液(LS緩衝液)に様々な濃度のSrCl2(0mM〜14mM)を添加した。ポリメラーゼ複合体は、洗浄緩衝液中で、わずか3.5mMのSr(最低濃度)の存在下においてもはるかに安定していた。さらに、結合工程中に正しいまたは誤ったヌクレオチドが存在する場合、複合体の安定性は顕著に影響されず、Srに関連するポリメラーゼの安定性が三元複合体に限定されるものではないことが示された。結果を図5に示す。
本実験では、DNA Pol Iの3′−5′エキソヌクレアーゼ活性の効果を調べた。ポリメラーゼのエキソヌクレアーゼ活性によって提供される校正機能が誤った塩基またはフラップを除去するため、エキソヌクレアーゼ活性は忠実度を向上させる。しかしながら、この活性は、潜在的に正しい塩基を切断して誤った配列読取り結果を導く可能性がある。3′末端にミスマッチを有するプライマーを鋳型にハイブリダイズさせて、ほつれ末端またはフラップ構築物を作製した。Klenow exo−ポリメラーゼはこの末端を伸長させることができない。しかしながら、DNA pol Iラージフラグメントはエキソヌクレアーゼ活性を有し、そしてミスマッチ塩基を除去することができ、一旦除去すると、klenow exo−ポリメラーゼまたはDNAポリメラーゼによってプライマーを正常に伸長することができる。
EML4−ALK融合は、非小細胞肺癌の患者の4〜5%に見出される(Soda et al.,Nature 448:561−6(2007);Mano,Cancer Sci.99:2349−55(2008);及びHorn and Pao,J.Clin.Oncol.27:4232−5(2009))。臨床肺腫瘍において、ALK C4493A突然変異が同定されており、この変異は、ALKタンパク質中にL1196M「ゲートキーパー」突然変異を生じさせ、化学療法薬であるクリゾチニブに対する耐性を付与する(Choi et al.,N.Engl.J.Med.18:1734−9(2010))。4496−4509ASプライマーは、ゲートキーパー突然変異を有する領域の配列決定を可能にする。鋳型オリゴヌクレオチド配列は、野生型ヒトALK遺伝子(ヌクレオチド番号4460−4509)に由来していた。プライマー配列は、ヒトALK遺伝子(ヌクレオチド番号4496−4509)に相補的であった。
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509SのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGG−3′− (3′−dT−5′)(配列番号9)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:5′−CCCGCCATGAGCTC−3′(配列番号10)
材料及び試薬。SPRセンサーチップ:20mm×20mm×1mm高屈折率(1.61)スライド(NanoSPR、シカゴ、イリノイ州)。アルカンチオール、PEG6:モノチオアルカン(C11)PEG6−OH(11−メルカプトウンデシルヘキサエチレングリコール(カタログ番号、SPT−0011P6);及びBAT:ビオチニル化アルカンPEGチオール(カタログ番号、SPT−0012D)は、Sensopath technologies(ボーズマン、モンタナ州)から入手した。塩基緩衝液(洗浄):300mM KCl、20mMトリスHCl(pH8.0)、0.01% Tween−20、1mM SrCl2、検査緩衝液:塩基緩衝液+50nM Klenow断片+100nM dNTP。組込み緩衝液:塩基緩衝液+10mM MgCl2。
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509SのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号9)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
オリゴヌクレオチド4460−4494ASのDNA配列:
5′−AGCAGGATGAACCGGG/i5NitInd/CAGGGATTGCAGGCTCAC−3′(配列番号11)、ここで、「/i5NitInd/」は5−ニトロインドール−2′−デオキシリボース残基である。5−ニトロインドールは、本文脈においては、グアニン四重鎖の形成を防止することを意図しており、ユニバーサル塩基として機能する。
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509SのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号9)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
オリゴヌクレオチド4460−4494ASのDNA配列:
5′−AGCAGGATGAACCGGG/i5NitInd/CAGGGATTGCAGGCTCAC−3′(配列番号11)、ここで「/i5NitInd/」は5−ニトロインドール−2′−デオキシリボース残基である。5−ニトロインドールは、本文脈においては、グアニン四重鎖の形成を防止することを意図しており、ユニバーサル塩基として機能する。
オリゴヌクレオチド4460−4494AS−T8のDNA配列:
5′−TTTTTTTTAGCAGGATGAACCGGG/i5NitInd/CAGGGATTGCAGGCTCAC−3′(配列番号12)、ここで「/i5NitInd/」は5−ニトロインドール−2′−デオキシリボース残基である。5−ニトロインドールは、本文脈においては、グアニン四重鎖の形成を防止することを意図しており、ユニバーサル塩基として機能する。
ssDNAプライマー/鋳型を調製するために、オリゴヌクレオチドBtn−4460−4509S(配列番号9)及び4496−4509AS(配列番号10)を、アニーリング緩衝液(10mMトリス pH8.0、0.1mM EDTA、80mM KCl)を含有するチューブ中で混合した。オリゴヌクレオチド溶液を含むチューブを乾燥ヒートブロック(95℃で5分間)にロードし、ブロックをベンチトップに移して周囲温度まで徐冷して鎖をアニーリングした。E.coli DNAポリメラーゼのKlenow(3′→5′exo−)断片は、Enzymatics(ビバリー、マサチューセッツ州;カタログ番号P7010−LC−L)から購入した。グルタミン酸カリウムは、(Teknova、ホリスター、カリフォルニア州;カタログ番号P2000)から購入した。超高純度ウシ血清アルブミン(BSA)はAmbion(フォスターシティ、カリフォルニア州)から購入した。すべての試薬及び溶液は分子生物学グレードであった。
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509SのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号9)
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509S C4493AのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGATGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号21)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
オリゴヌクレオチド4460−4494AS−T8のDNA配列:
5′−TTTTTTTTAGCAGGATGAACCGGG/i5NitInd/CAGGGATTGCAGGCTCAC−3′(配列番号12)、ここで「/i5NitInd/」は5−ニトロインドール−2′−デオキシリボース残基である。5−ニトロインドールは、本文脈においてグアニン四重鎖の形成を防止することを意図しており、ユニバーサル塩基として機能する。
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509S C4493AのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGATGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号21)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
鋳型オリゴヌクレオチドphiX_100mismatchのDNA配列:
ビオチン−5′−GGCAAATCACCAGAAGGCGGTTCCTGAATGAATGGGAAGCCTTCAAGAA−GGTGATAAGCAGGAGAAACATACGAAGCATCATAACGATACCACTGACCC−3′(配列番号22)
プライマーオリゴヌクレオチドFP2のDNA配列:
5′−GAGGGTCAGTGGTATCGTTATG−3′(配列番号5)
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509S C4493AのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGATGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号21)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
3′逆位dTを有する鋳型オリゴヌクレオチドBtn−4460−4509S C4493AのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGATGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号21)
プライマーオリゴヌクレオチド4496−4509ASのDNA配列:
5′−CCCGCCATGAGCTC−3′(配列番号10)
3′逆位dTを有する野生型ALK鋳型オリゴヌクレオチドBtn−4460−4509SのDNA配列:
ビオチン−5′−GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGG−3′−(3′−dT−5′)(配列番号7)
5′−CCCGCCATGAGCTCCA−3′(配列番号26)
ALK−G2プライマーオリゴヌクレオチド4491−4509ASのDNA配列:
5′−CCCGCCATGAGCTCCAGCA−3′(配列番号27)
ALK−G3プライマーオリゴヌクレオチド4476−4509ASのDNA配列:
5′−CCCGCCATGAGCTCCAGCAGGATGAACC/ideoxyI/GGGCA−3′(配列番号28)、ここで、「/ideoxyI/」は、2′−デオキシイノシン残基である。
ALK−G4プライマーオリゴヌクレオチド4482−4509ASのDNA配列:
5′−CCCGCCATGAGCTCCAGCAGGATGAACC−3′(配列番号29)
Claims (23)
- 鋳型核酸分子の配列決定方法であって:
(a)プライマーでプライミングした鋳型核酸分子を提供し;
(b)前記プライミングした鋳型核酸分子を、ポリメラーゼ及び第一の非標識ヌクレオチド分子を含む第一の反応混合物と接触させ、
そこにおいて、前記第一の非標識ヌクレオチド分子が前記プライミングした鋳型核酸分子の後続塩基に相補的である場合、前記プライミングした鋳型核酸分子、前記ポリメラーゼ及び前記第一の非標識ヌクレオチド分子が、三元複合体を形成することができ、
前記接触は、前記プライミングした鋳型核酸分子、前記第一の非標識ヌクレオチド分子及び前記ポリメラーゼ間の、三元複合体の形成を安定化する条件下で行い;
(c)前記ポリメラーゼ及び前記プライミングした鋳型核酸分子間で形成された任意の二元複合体を除去する洗浄工程を実施し;
(d)前記第一の非標識ヌクレオチド分子の存在下で、前記プライミングした鋳型核酸分子の前記プライマーへの前記第一の非標識ヌクレオチド分子の化学的組込みを伴わずに、前記ポリメラーゼと、前記プライミングした鋳型核酸分子との相互作用をモニタリングし;ならびに
(e)工程(d)の前記モニタリングした相互作用によって、前記プライミングした鋳型核酸分子の前記後続塩基に相補的なヌクレオチドを同定することを含む検査工程を含み、
工程(b)及びモニタリング工程(d)が触媒金属イオンの非存在下で行われる、前記配列決定方法。 - 工程(b)、(c)及び(d)を1回以上繰り返すことをさらに含む、請求項1に記載の方法。
- 工程(b)を、前記ポリメラーゼ及び第二の非標識ヌクレオチド分子を含む第二の反応混合物を使用して繰り返す、請求項2に記載の方法。
- 前記ポリメラーゼ及び第三の非標識ヌクレオチド分子を含む第三の反応混合物を用いて工程(b)を繰り返す工程をさらに含む、請求項3に記載の方法。
- 前記ポリメラーゼ及び第四の非標識ヌクレオチド分子を含む第四の反応混合物を用いて工程(b)を繰り返す工程をさらに含む、請求項4に記載の方法。
- 前記第一の反応混合物が、前記三元複合体の形成を安定化する非触媒金属イオンを含む、請求項1〜5のいずれか一項に記載の方法。
- 前記条件が安定化剤を含み、かつ前記安定化剤が非触媒金属イオンである、請求項1〜6のいずれか一項に記載の方法。
- 前記非触媒金属イオンが、ストロンチウム、スズ、またはニッケルである、請求項6または7に記載の方法。
- 前記三元複合体が半減期を有し、非標識ヌクレオチド分子が前記プライミングした鋳型核酸分子の前記後続塩基に相補的な塩基を提供する場合に形成する前記三元複合体の前記半減期よりも短い期間、前記洗浄工程を実施する、請求項1〜8のいずれか一項に記載の方法。
- 前記ポリメラーゼがプロセッシブポリメラーゼである、請求項1〜9のいずれか一項に記載の方法。
- 前記ポリメラーゼがディストリビューティブポリメラーゼである、請求項1〜9のいずれか一項に記載の方法。
- 工程(e)の後にリロード工程をさらに含み、前記リロード工程が、前記三元複合体を安定化させる条件下で、前記プライミングした鋳型核酸と、前記ポリメラーゼ及び第一の非標識ヌクレオチド分子を含むリロード混合物とを接触させる工程を含む、請求項1〜11のいずれか一項に記載の方法。
- 工程(e)の後に組込み工程をさらに含み、前記組込み工程が、前記プライミングした核酸分子の前記後続塩基に相補的な前記非標識ヌクレオチドを前記プライマーに組込む工程を含む、請求項1〜12のいずれか一項に記載の方法。
- 前記組込みが、組込み反応混合物と接触させることを含み、前記組込み反応混合物が、前記プライミングした鋳型核酸分子の前記プライマーに組込み可能な1つ以上の非標識ヌクレオチド分子及び組込み不可能な1つ以上の非標識ヌクレオチド分子を含む、請求項13に記載の方法。
- モニタリング工程(d)を、表面プラズモン共鳴センサー上で実行する、請求項1〜14のいずれか一項に記載の方法。
- 前記プライマーでプライミングした鋳型核酸分子を、前記表面プラズモン共鳴センサー上に配置する、請求項15に記載の方法。
- モニタリング工程(d)を、前記ポリメラーゼからの固有シグナルの検出を介して行う、請求項1〜16のいずれか一項に記載の方法。
- 前記ポリメラーゼが非標識ポリメラーゼであり、モニタリング工程(d)を、前記ポリメラーゼに付随する検出可能な標識の非存在下で行う、請求項1〜17のいずれか一項に記載の方法。
- モニタリング工程(d)が、前記第一の非標識ヌクレオチド分子の存在下で、前記プライミングした鋳型核酸分子の前記プライマーへの前記第一の非標識ヌクレオチド分子の化学的組込みを伴わずに、前記ポリメラーゼと前記プライミングした鋳型核酸分子との定常状態相互作用をモニタリングする工程を含む、請求項1〜18のいずれか一項に記載の方法。
- 前記プライマーが、ブロッキング部分を含む、請求項1〜19のいずれか一項に記載の方法。
- 前記第一の反応混合物がポリメラーゼ阻害剤を含む、請求項1〜20のいずれか一項に記載の方法。
- 前記非標識ヌクレオチド分子がブロッキング部分を含む、請求項1〜21のいずれか一項に記載の方法。
- 前記ポリメラーゼが検出可能なタグを含む、請求項1〜22のいずれか一項に記載の方法。
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