JP4177118B2 - 増幅方法 - Google Patents
増幅方法 Download PDFInfo
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- JP4177118B2 JP4177118B2 JP2002585667A JP2002585667A JP4177118B2 JP 4177118 B2 JP4177118 B2 JP 4177118B2 JP 2002585667 A JP2002585667 A JP 2002585667A JP 2002585667 A JP2002585667 A JP 2002585667A JP 4177118 B2 JP4177118 B2 JP 4177118B2
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- JP
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- Prior art keywords
- ppase
- pyrophosphate
- reaction mixture
- pcr
- polymerase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 239000011706 ferric diphosphate Substances 0.000 description 1
- 235000007144 ferric diphosphate Nutrition 0.000 description 1
- CADNYOZXMIKYPR-UHFFFAOYSA-B ferric pyrophosphate Chemical compound [Fe+3].[Fe+3].[Fe+3].[Fe+3].[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O CADNYOZXMIKYPR-UHFFFAOYSA-B 0.000 description 1
- 229940036404 ferric pyrophosphate Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007849 hot-start PCR Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 150000002843 nonmetals Chemical class 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- WRMXOVHLRUVREB-UHFFFAOYSA-N phosphono phosphate;tributylazanium Chemical compound OP(O)(=O)OP([O-])([O-])=O.CCCC[NH+](CCCC)CCCC.CCCC[NH+](CCCC)CCCC WRMXOVHLRUVREB-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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Description
(DNA)n残基+dNTP⇔(DNA)n+1残基+PPi
例えばDNAシーケンシング反応で高濃度のPPiが存在すると上記反応は逆方向になることが知られている。これはピロリン酸分解として知られており、熱安定性DNAポリメラーゼを使用して70℃でDNAシーケンシングする場合に問題となることが認められている。DNAシーケンシングで使用するDNAポリメラーゼ製剤に熱安定性PPaseを加えることによりこの問題は解決された。
Taq DNAポリメラーゼを使用し、無機ピロリン酸塩として各種量のピロリン酸四ナトリウム・10水和物(PPi)の存在下に下記試薬を使用して標準の500bpのラムダ鋳型を用いるPCRを実施した。
i)94℃で3.00分間、
ii)94℃で10秒間、50℃で10秒間、72℃で30秒間を20サイクル、
iii)72℃で7分間、
iv)25℃に保温。
1+2 0 PPi
3+4 1mM PPi
5+6 2mM PPi
7+8 3mM PPi
9+10 4mM PPi
11 5mM PPi。
MgはPPiに結合するので、実施例1の結果はMgが過剰のPPiによりキレート化されたためであるとも考えられる。その結果、プライマー伸長を進行させるにはMgが不足したとも考えられる。この可能性を排除するために、各種濃度のマグネシウムイオンの存在下に3mM PPiを使用して実施例1の手順を繰返した。
1+2 1.5mM MgCl2
3+4 5mM MgCl2
5+6 7.5mM MgCl2
7+8 10mM MgCl2
9+10 1.5mM MgCl2+3mM PPi
11+12 5mM MgCl2+3mM PPi
13+14 7.5mM MgCl2+3mM PPi
15 10mM MgCl2+3mM PPi。
ピロリン酸塩(PPi)を加えた反応混合物にSulfolobus acidocaldarius PPase(Sac PPase)0.2uを加えた以外は実施例1の500bpラムダPCRを繰返した。反応混合物をSac PPase 0.2uの存在下で95℃に5分間保温すると、ピロリン酸塩を分解してPCR反応を進行させるに十分であった。
上段
1+2 1mM PPi+0.2u PPase
3+4 2mM PPi+0.2u PPase
5+6 3mM PPi+0.2u PPase
7+8 4mM PPi+0.2u PPase
9+10 5mM PPi+0.2u PPase
下段
1+2 1mM PPi
3+4 2mM PPi
5+6 3mM PPi
7+8 4mM PPi
9+10 5mM PPi
11+12 0mM PPi。
次に、正しい寸法のPCR産物を生産するために「ホットスタート」反応を必要とするアッセイシステムに本発明の方法を適用した。
i)95℃で2.00分以下、
ii)95℃で15秒間、50℃で30秒間、72℃で30秒間を35サイクル、
iii)72℃で7分間、
iv)25℃に保温。
1 標準TaqポリメラーゼPCR,ベタイン無,ミスプライミング多数;
2 標準TaqポリメラーゼPCR,ベタイン有,明バンドが正しい産物,多少ミスプライミング有;
3 標準TaqポリメラーゼPCR,ベタイン無,3mM PPiと0.2u Sac PPase添加,ミスプライミング皆無,95℃で5分間変性;
4 標準TaqポリメラーゼPCR,ベタイン有,3mM PPiと0.2u Sac PPase添加,正しい産物のみ,95℃で5分間変性;
5+6 95℃で2分間変性以外は3と同じ;
7+8 95℃で2分間変性以外は4と同じ。
0.2u Sac PPaseと3mM PPiを加えたPCR混合物をアンギオテンシンアッセイ実施前に各種時間20℃の室温に放置した場合の影響を調べた。Sac PPaseは熱安定性酵素であるが、室温で低レベルの酵素活性がある可能性があった。その結果、DNAポリメラーゼを抑制/停止させるには反応にPPiが不足し、プライマー伸長が起こり、「ホットスタート」機能が失われる恐れがあった。
Sac PPaseの代わりに別の市販熱安定性PPase(活性単位の定義は異なる)を使用して同様の反応を行い、実施例4に記載したアッセイを繰返した。結果を図6に示す。同図中、各レーンは以下の反応を表す。
1+2 標準TaqポリメラーゼPCR,ベタイン無;
3+4 標準TaqポリメラーゼPCR,ベタイン有;
5+6 標準TaqポリメラーゼPCR,ベタイン無,3mM PPiと0.2u Sac PPase添加;
7+8 標準TaqポリメラーゼPCR,ベタイン有,3mM PPiと0.2u Sac PPase添加;
9+10 標準TaqポリメラーゼPCR,ベタイン無,3mM PPiと10u*Thermococcus litoralis PPase添加;
11+12 標準TaqポリメラーゼPCR,ベタイン有,3mM PPiと10u*Thermococcus litoralis PPase添加。
本発明の方法と数種の比較アッセイで各種熱安定性DNAポリメラーゼを使用した。使用したポリメラーゼは数種の非プルーフリーディングThermus種DNAポリメラーゼ、プルーフリーディング超好熱始原菌DNAポリメラーゼ、及び非プルーフリーディングDNAポリメラーゼとプルーフリーディングDNAポリメラーゼの混合物である。
レーン
上段
1+2 Taqポリメラーゼ,0mM PPi,PPase無;
3+4 Taqポリメラーゼ,3mM PPi,PPase無;
5+6 Taqポリメラーゼ,3mM PPi,0.2u Sac PPase;
7+8 Tbrポリメラーゼ,0mM PPi,PPase無;
9+10 Tbrポリメラーゼ,3mM PPi,PPase無;
11+12 Tbrポリメラーゼ,3mM PPi,0.2u Sac PPase;
下段
1+2 Tthポリメラーゼ,0mM PPi,PPase無;
3+4 Tthポリメラーゼ,3mM PPi,PPase無;
5+6 Tthポリメラーゼ,3mM PPi,0.2u Sac PPase;
7+8 TspNHポリメラーゼ,0mM PPi,PPase無;
9+10 TspNHポリメラーゼ,3mM PPi,PPase無;
11+12 TspNHポリメラーゼ,3mM PPi,0.2u Sac PPase。
レーン
上段
1+2 Pfuポリメラーゼ,0mM PPi,PPase無;
3+4 Pfuポリメラーゼ,3mM PPi,PPase無;
5+6 Pfuポリメラーゼ,3mM PPi,0.2u Sac PPase;
7+8 9°Nエキソポリメラーゼ,0mM PPi,PPase無;
9+10 9°Nエキソポリメラーゼ,3mM PPi,PPase無;
11+12 9°Nエキソポリメラーゼ,3mM PPi,0.2u Sac PPase;
下段
1+2 VENTポリメラーゼ,0mM PPi,PPase無;
3+4 VENTポリメラーゼ,3mM PPi,PPase無;
5+6 VENTポリメラーゼ,3mM PPi,0.2u Sac PPase。
レーン
1+2 Taqポリメラーゼ,ベタイン無;
3+4 Taqポリメラーゼ,ベタイン有;
5+6 Accuraseポリメラーゼ,ベタイン無;
7+8 Accuraseポリメラーゼ,ベタイン有;
9+10 Tbrポリメラーゼ,ベタイン無;
11+12 Tbrポリメラーゼ,ベタイン有;
13+14 Tthポリメラーゼ,ベタイン無;
15+16 Tthポリメラーゼ,ベタイン有。
対照レーン1〜4(上段)及び12〜16(下段)
レーン
上段
1+2 Taqポリメラーゼ,ベタイン無,3mM PPi,Sac PPase無;
3+4 Taqポリメラーゼ,ベタイン有,3mM PPi,Sac PPase無;
以下、全て3mM PPiと0.2u Sac PPase添加
5+6 Taqポリメラーゼ,ベタイン無;
7+8 Taqポリメラーゼ,ベタイン有;
9+10 Accuraseポリメラーゼ,ベタイン無;
11+12 Accuraseポリメラーゼ,ベタイン有;
13+14 Tbrポリメラーゼ,ベタイン無;
15+16 Tbrポリメラーゼ,ベタイン有。
下段
以下、全て3mM PPiと0.2u Sac PPase添加
1+2 TthポリメラーゼPCR,ベタイン無;
3+4 TthポリメラーゼPCR,ベタイン有;
5+6 TspNHポリメラーゼ,ベタイン無;
7+8 TspNHポリメラーゼ,ベタイン有;
9+10 Pfuポリメラーゼ,ベタイン無;
11+12 Pfuポリメラーゼ,ベタイン有;
13+14 Taqポリメラーゼ対照,ベタイン無,PPi又はPPase無;
15+16 Taqポリメラーゼ対照,ベタイン有,PPi又はPPase無。
当初の結果(図9及び10)によると、化学修飾Taqポリメラーゼ(米国特許第5,677,152号に記載されているように修飾)はベタインの不在下では多少の偽PCR産物を生産するが、ベタインの存在下では正しい産物が得られる。
レーン
1+2 Taqポリメラーゼ,ベタイン無;
3+4 Taqポリメラーゼ,ベタイン有;
5+6 化学修飾Taq,ベタイン無;
7+8 化学修飾Taq,ベタイン有;
9+10 本発明の方法(3mM PPiと2u Sac PPase),ベタイン無;
11+12 本発明の方法(3mM PPiと2u Sac PPase),ベタイン有。
レーン
1+2 抗Taq抗体+Taqポリメラーゼ,ベタイン無;
3+4 抗Taq抗体+Taqポリメラーゼ,ベタイン有。
微生物系統保存施設のカルチャーコレクションからAeropyrum pernixを入手した。無機ピロホスファターゼ酵素をクローニングし、発現させ、精製した。
Aeropyrum pernixのピロホスファターゼ遺伝子を含むゲノム配列を図11に示す。使用したプライマーはAeropyrum pernixのゲノム配列から設計した。これらを5’→3’ として以下に示し、制限部位を太字で示す。
94℃で3分間初期保温、
94℃で10秒間、55℃で10秒間、68℃で45秒間を20サイクル、
72℃で7分間最終保温。
50pM上流プライマー(5’..TGCATGCATATGACAGGCTGTCTGAAAATTG..3’−配列番号18)
50pM下流プライマー(5’..TAAGTGTAAGCTTGACTGTGGGGGCGGTGAAAG..3’−配列番号19)
1.5mM MgCl2
1.25u Accurase DNAポリメラーゼ(カタログ番号AC001,GeneSys Ltd.)
75mM Tris,pH8.8
20mM硫酸アンモニウム
0.1%(w/v)Tween 20
100ng Aeropyrum pernixゲノムDNA。
このクローンをLB24リットルに移した。OD600が約1.5に達したら、0.5mM IPTGを加えて培養を誘導し、更に4時間増殖させた。次に細胞を回収し、細胞ペレットを溶解させた。発現酵素をフェニルセファロースCL4B(Amersham Pharmacia Biotech)、ヒドロキシアパタイト(Bio−rad Laboratories)及び高性能Qセファロース(Amersham Pharmacia Biotech)で標準カラムクロマトグラフィ¬により精製し、最後に20mM Tris−HCl,pH8.0,100mM NaCl,0.5%(v/v)Tween 20,0.5%(v/v)Nonidet P40,0.1mM EDTA,1mMジチオスレイトール及び50%グリセロール中で−20℃で保存した。
A.pernix無機ピロホスファターゼ酵素を使用して本発明の方法を実施した。このアッセイの基本はヒトBアクチン遺伝子の増幅である。
1倍反応緩衝液
dATP、dCTP、dGTP各200μM及びdUTP400μM
0.025u/μl未修飾Taqポリメラーゼ
0.002u/μl Aeropyrum pernix無機ピロホスファターゼ
0.3μM 5’プライマー(5’GAC TCG TCA TAC TCC TGC TTG CT 3’−配列番号22)
0.3μM 3’プライマー(5’CAT TGC CGA CAG GAT GCA GAA 3’−配列番号23)
0.15μM Taqmanプローブ(FAM−ATCCACATCTGCTGGAAGGTGGACAGT−TAMRA−配列番号24)
5mM MgCl2
2mM NaPPi
受動的参照
7.5ngから出発したヒトゲノムDNAの4倍希釈液(2500コピー)。
94℃で3分間初期変性、
94℃で15秒間と60℃で60秒間を40サイクル。
Claims (23)
- 核酸増幅反応を実施するための方法であって、該方法は、
ポリメラーゼ連鎖反応によって増幅反応を実施するために必要な試薬と、プライマー伸長を妨げる量のピロリン酸塩が混合された、PCR反応混合物を形成する段階、
該混合物にPCR反応混合物50μLあたり少なくとも0.04単位の熱安定性ピロホスファターゼ酵素(PPase)を加える段階、及び
該ピロリン酸塩がピロホスファターゼ酵素(PPase)により消化される条件下に該反応混合物をおくことによって増幅反応が実施される段階を含む前記方法。 - 核酸増幅反応を実施するための方法であって、該方法は、
PCR増幅反応を実施するために必要な試薬と、プライマー伸長を妨げる量のピロリン酸塩が混合された、PCR反応混合物を形成する段階、
該混合物に超好熱始原菌から得られる熱安定性ピロホスファターゼ酵素(PPase)を加える段階、及び
該ピロリン酸塩がピロホスファターゼ酵素(PPase)により消化される条件下に該反応混合物をおくことにより増幅反応が実施される段階を含む前記方法。 - 反応混合物がサーマス アクアティカス(Thermus aquaticus)ポリメラーゼ(Taq)、サーマス サーモフィラス(Thermus thermophilus)ポリメラーゼ(Tth)、サーマス(Thermus)種NHポリメラーゼ(TspNH)、サーマス ブロキアヌス(Thermus brockianus)ポリメラーゼ(Tbr)、ピロコッカス フリオサス(Pyrococcus furiosus)ポリメラーゼ(Pfu)、9°N7 エキソDNAポリメラーゼ、及びサーモコッカス リトラリス(Thermococcus litoralis) DNAポリメラーゼから選択されるDNAポリメラーゼを含む請求項1又は2に記載の方法。
- ピロリン酸塩がアルカリ金属ピロリン酸塩である請求項1から3のいずれか一項に記載の方法。
- ピロリン酸塩が式Na4P2O7のピロリン酸四ナトリウムである請求項4に記載の方法。
- ピロリン酸塩が少なくとも0.5mMの濃度で反応混合物中に存在する請求項1から5のいずれか一項に記載の方法。
- ピロリン酸塩が1〜10mMの濃度で存在する請求項6に記載の方法。
- 熱安定性PPaseがスルホロバス アシドカルダリウス(Sulfolobus acidocaldarius)無機ピロホスファターゼ(Sac PPase)、サーモコッカス リトラリス(Thermococcus litoralis)無機ピロホスファターゼ又はアエロピラム ペルニクス(Aeropyrum pernix)無機ピロホスファターゼである請求項1から7のいずれか一項に記載の方法。
- 熱安定性PPaseが配列番号25に示すアミノ酸配列又はその変異体もしくはフラグメントを含む請求項1から8のいずれか一項に記載の方法。
- 熱安定性PPaseを反応混合物の形成直後に添加する請求項8又は9に記載の方法。
- 存在するピロリン酸塩をPPaseで消化するために増幅反応前に50℃以上の温度でインキュベーションする段階を含む請求項10に記載の方法。
- PPaseをPCR反応混合物50μL当たり少なくとも0.04uの濃度で反応混合物に加える請求項2に記載の方法。
- PPaseをPCR反応混合物50μL当たり0.08uの濃度で反応混合物に加える請求項1から12のいずれか一項に記載の方法。
- PPaseをPCR反応混合物50μL当たり0.2〜10uの濃度で反応混合物に加える請求項12又は13に記載の方法。
- 増幅反応を実施するためのキットであって、ピロリン酸塩と、PCR反応混合物50μL当たり少なくとも0.04uとなる量の熱安定性PPaseを含む前記キット。
- 増幅反応で使用するのに必要な1種以上の試薬を含む請求項15に記載のキット。
- 特定ターゲット核酸の増幅を実施するために必要な1種以上のプライマーを更に含む請求項16に記載のキット。
- 1種以上の蛍光標識試薬を更に含む請求項15から17のいずれか一項に記載のキット。
- 蛍光標識試薬がインターカレーター色素、蛍光標識プローブ、蛍光標識プライマー又は蛍光標識ヌクレオチドの1種以上から選択される請求項18に記載のキット。
- 請求項1から14のいずれか一項に記載の増幅反応を実施するための方法における熱安定性ピロホスファターゼ酵素の使用。
- ピロホスファターゼ酵素がアエロピラム ペルニクス(Aeropyrum pernix)から単離した酵素である請求項20に記載の使用。
- ピロホスファターゼ酵素が配列番号26に示すポリヌクレオチド配列又はその変異体もしくはフラグメントによりコードされる請求項20に記載の使用。
- ピロホスファターゼ酵素が配列番号25に示すアミノ酸配列又はその変異体もしくはフラグメントを含む請求項20に記載の使用。
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