JP4643263B2 - 対立遺伝子特異的プライマー伸長 - Google Patents
対立遺伝子特異的プライマー伸長 Download PDFInfo
- Publication number
- JP4643263B2 JP4643263B2 JP2004532003A JP2004532003A JP4643263B2 JP 4643263 B2 JP4643263 B2 JP 4643263B2 JP 2004532003 A JP2004532003 A JP 2004532003A JP 2004532003 A JP2004532003 A JP 2004532003A JP 4643263 B2 JP4643263 B2 JP 4643263B2
- Authority
- JP
- Japan
- Prior art keywords
- phosphate
- dna polymerase
- labeled
- nucleic acid
- primer
- 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.)
- Expired - Lifetime
Links
- 108700028369 Alleles Proteins 0.000 title claims description 22
- 238000000034 method Methods 0.000 claims description 98
- 125000003729 nucleotide group Chemical group 0.000 claims description 92
- 239000002773 nucleotide Substances 0.000 claims description 90
- 238000006243 chemical reaction Methods 0.000 claims description 86
- 150000007523 nucleic acids Chemical class 0.000 claims description 74
- 229910019142 PO4 Inorganic materials 0.000 claims description 72
- 239000010452 phosphate Substances 0.000 claims description 70
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 69
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 69
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 59
- 229920000388 Polyphosphate Polymers 0.000 claims description 56
- 239000001205 polyphosphate Substances 0.000 claims description 56
- 235000011176 polyphosphates Nutrition 0.000 claims description 56
- 102000039446 nucleic acids Human genes 0.000 claims description 52
- 108020004707 nucleic acids Proteins 0.000 claims description 52
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 29
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 29
- 238000001514 detection method Methods 0.000 claims description 27
- 239000013626 chemical specie Substances 0.000 claims description 22
- -1 Amplitaq FS Proteins 0.000 claims description 20
- 101710163270 Nuclease Proteins 0.000 claims description 15
- 230000000295 complement effect Effects 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 10
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 10
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 10
- 235000000346 sugar Nutrition 0.000 claims description 10
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- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 229910052717 sulfur Chemical group 0.000 claims description 7
- 108010017826 DNA Polymerase I Proteins 0.000 claims description 6
- 102000004594 DNA Polymerase I Human genes 0.000 claims description 6
- 125000002015 acyclic group Chemical group 0.000 claims description 6
- 125000002837 carbocyclic group Chemical group 0.000 claims description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 claims description 4
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- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 4
- IXZONVAEGFOVSF-UHFFFAOYSA-N 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone Chemical group OP(O)(=O)OC1=CC=C(Cl)C=C1C1=NC(=O)C2=CC(Cl)=CC=C2N1 IXZONVAEGFOVSF-UHFFFAOYSA-N 0.000 claims description 3
- 125000000824 D-ribofuranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@]1([H])O[H] 0.000 claims description 3
- BVTJGGGYKAMDBN-UHFFFAOYSA-N Dioxetane Chemical class C1COO1 BVTJGGGYKAMDBN-UHFFFAOYSA-N 0.000 claims description 3
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- HSSLDCABUXLXKM-UHFFFAOYSA-N resorufin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3N=C21 HSSLDCABUXLXKM-UHFFFAOYSA-N 0.000 claims description 3
- IMZBXSAKACGTPH-UHFFFAOYSA-N (3-oxo-6'-phosphonooxyspiro[2-benzofuran-1,9'-xanthene]-3'-yl) dihydrogen phosphate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OP(O)(O)=O)C=C1OC1=CC(OP(O)(=O)O)=CC=C21 IMZBXSAKACGTPH-UHFFFAOYSA-N 0.000 claims description 2
- FBMZEITWVNHWJW-UHFFFAOYSA-N 1,7-dihydropyrrolo[2,3-d]pyrimidin-4-one Chemical compound OC1=NC=NC2=C1C=CN2 FBMZEITWVNHWJW-UHFFFAOYSA-N 0.000 claims description 2
- JTNGEYANGCBZLK-UHFFFAOYSA-N 1h-indol-3-yl dihydrogen phosphate Chemical compound C1=CC=C2C(OP(O)(=O)O)=CNC2=C1 JTNGEYANGCBZLK-UHFFFAOYSA-N 0.000 claims description 2
- UMTZYGZMIJRCFG-UHFFFAOYSA-N 3-phenyldioxetane Chemical compound C1OOC1C1=CC=CC=C1 UMTZYGZMIJRCFG-UHFFFAOYSA-N 0.000 claims description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 claims description 2
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 claims description 2
- 229930024421 Adenine Natural products 0.000 claims description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims description 2
- 102100034343 Integrase Human genes 0.000 claims description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 2
- 229960000643 adenine Drugs 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 238000004040 coloring Methods 0.000 claims description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 claims description 2
- 150000008163 sugars Chemical class 0.000 claims description 2
- 125000004434 sulfur atom Chemical group 0.000 claims description 2
- 150000007970 thio esters Chemical class 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 2
- 229940113082 thymine Drugs 0.000 claims description 2
- 229940035893 uracil Drugs 0.000 claims description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 claims 1
- WSROMHLXVLTYTH-UHFFFAOYSA-N (9,9-dimethyl-7-oxoacridin-2-yl) dihydrogen phosphate Chemical compound C1=C(OP(O)(O)=O)C=C2C(C)(C)C3=CC(=O)C=CC3=NC2=C1 WSROMHLXVLTYTH-UHFFFAOYSA-N 0.000 claims 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 claims 1
- 108010006785 Taq Polymerase Proteins 0.000 claims 1
- YACKEPLHDIMKIO-UHFFFAOYSA-L methylphosphonate(2-) Chemical compound CP([O-])([O-])=O YACKEPLHDIMKIO-UHFFFAOYSA-L 0.000 claims 1
- 125000004430 oxygen atom Chemical group O* 0.000 claims 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 claims 1
- 239000013615 primer Substances 0.000 description 107
- 235000021317 phosphate Nutrition 0.000 description 55
- 239000000523 sample Substances 0.000 description 25
- 241000894007 species Species 0.000 description 25
- 108091028043 Nucleic acid sequence Proteins 0.000 description 22
- 239000002777 nucleoside Substances 0.000 description 17
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- 239000000047 product Substances 0.000 description 11
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
- 235000011180 diphosphates Nutrition 0.000 description 9
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 3
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
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Description
ハイブリッドは以下のように形成できる。
δ−9H−(1,3−ジクロロ−9,9−ジメチルアクリジン−2−オン−7−イル)−チミジン−5′−四リン酸(dT4P−DDAO)及び色素結合四リン酸の調製
TTP TEA塩10μモルを蒸発乾固した。残渣にトリブチルアミン40μモル及び乾燥ピリジン5mlを添加した。この溶液を再度蒸発乾固した。乾燥ジメチルホルムアミド3mlで2回、共蒸発した後、残渣を乾燥DMF200μlに再溶解し、アルゴンを流し込み、栓をした。シリンジを用いて、乾燥DMF100μlに溶かしたカルボニルジイミダゾール(CDI)50μモル(8mg)を添加した。フラスコを外界温度で4時間撹拌した。
対立遺伝子特異的プライマー及び末端リン酸標識ヌクレオシドポリリン酸の識別
以下のプライマー及び鋳型を実施例2、3及び4で使用した。
プライマー:
5′−GTT CTC GGC ATC ACC ATC CG(s)T−3′(配列番号9)
5′−GTT CTC GGC ATC ACC ATG CG(s)T−3′(配列番号10)
5′−GTT CTC GGC ATC ACC ATC GG(s)T−3′(配列番号11)
鋳型:
5′−CAC CCT TAT CTG GTT GTC GAC GGA TGG TGA TGC CGA GAA C−3′(#1,配列番号12)
5′−CAC CCT TAT CTG GTT GTC GGC GGA TGG TGA TGC CGA GAA C−3′(#2,配列番号13)
5′−CAC CCT TAT CTG GTT GTC GCC GGA TGG TGA TGC CGA GAA C−3′(#3,配列番号14)
5′−CAC CCT TAT CTG GTT GTC GTC GGA TGG TGA TGC CGA GAA C−3′(#4,配列番号15)
反応は実施例(1)のデオキシヌクレオチドを用いて、室温(23℃)で構築した。反応液は、異なる2種のオリゴヌクレオチドの一方にアニールする単一のオリゴヌクレオチドプライマー(配列番号9で示される)を有するプライマー鋳型の組合せを含んでいた;2種の鋳型はプライマーの3′末端ヌクレオチドの反対側の多形部位にdA又はdGのいずれかを有し、それぞれ鋳型#1及び鋳型#2に相当するものである。
対立遺伝子特異的プライマーと末端リン酸標識ヌクレオシドポリリン酸を用いる対立遺伝子の識別:内部ミスマッチの影響
図4aについて、配列番号9の配列をもつプライマー及び3′末端に正しい又は間違った塩基をもつ鋳型#1又は鋳型#2を使用した。図4bについて、3′末端から3塩基離れて内部ミスマッチをもつ配列番号10のプライマー及び3′末端に正しい又は間違った塩基をもつ鋳型#1又は鋳型#2を使用した。図7は本発明の実施例3に使用したアニールしたプライマー/鋳型対を示す模式図である。
対立遺伝子特異的プライマーと末端リン酸標識ヌクレオシドポリリン酸を用いるPhi29DNAポリメラーゼによる対立遺伝子の識別
プライマーの3′末端に正しい又は間違った塩基をもち、同時に内部ミスマッチをもつ実施例2及び3に報告した実験を、Sequenaseの代わりにPhi29D12ADNAポリメラーゼを使用して繰返した。
Claims (16)
- 鋳型核酸の多形部位を検出する方法であって、当該方法が、
(a)多形部位を含む鋳型核酸と、対立遺伝子特異的プライマーであってその3′末端塩基が上記鋳型核酸の多形部位のヌクレオチドと相補的である対立遺伝子特異的プライマーと、式B−S−Y−(P)n−P−L(式中、Pはリン酸(PO3)であり、nは3以上であり、Yは酸素又はイオウ原子であり、Bは含窒素複素環式塩基であり、Sは非環式部分、炭素環式部分又は糖部分であり、Lは、天然又は修飾ヌクレオチドの末端リン酸でのリン酸エステル、チオエステル又はホスホルアミデート結合の形成に適したヒドロキシル基、スルフヒドリル基又はアミノ基を含有する酵素活性化可能標識である。)の2種以上の末端リン酸標識ヌクレオチドと、DNAポリメラーゼとを含む溶液中で核酸ポリメラーゼ反応を実施して、標識ポリリン酸を生成させる段階、
(b)段階(a)の溶液をホスファターゼと混合して、上記標識ポリリン酸から検出可能な標識化学種を生成させる段階、及び
(c)検出可能な標識化学種を検出する段階
を含んでおり、前記末端リン酸標識ヌクレオチドにおける酵素活性化可能標識が、化学発光化合物、蛍光発生色素及び発色色素からなる群から選択される標識であり、標識化学種の検出が、上記鋳型核酸における多形部位の存在を示す、方法。 - 前記段階(a)と段階(b)を同時に実施する、請求項1記載の方法。
- 前記対立遺伝子特異的プライマーがヌクレアーゼ耐性であり、前記段階(a)における核酸ポリメラーゼ反応がさらに3′→5′エキソヌクレアーゼ活性を有する酵素を含む、請求項1又は請求項2記載の方法。
- 前記ヌクレアーゼ耐性プライマーが、その3′末端にホスホン酸メチル、ボラノホスフェート又はホスホロチオエート結合を有する、請求項3記載の方法。
- 前記標識ポリリン酸とは異なる1種以上の追加の検出試薬を前記段階(a)の反応に添加する、請求項1乃至請求項3のいずれか1項記載の方法。
- 前記対立遺伝子特異的プライマーがその3′末端塩基から1、2又は3個目の塩基にミスマッチを有する、請求項1乃至請求項5のいずれか1項記載の方法。
- 前記DNAポリメラーゼがDNAポリメラーゼIのクレノーフラグメント、Phi29DNAポリメラーゼ、DNAポリメラーゼI、T4DNAポリメラーゼ、Thermo Sequenase、Sequenase、Taq DNAポリメラーゼ、pfu DNAポリメラーゼ、Amplitaq FS、逆転写酵素及びT7 DNAポリメラーゼからなる群から選択される、請求項1記載の方法。
- 前記DNAポリメラーゼが3′→5′エキソヌクレアーゼ活性を有し、前記対立遺伝子特異的プライマーがヌクレアーゼ耐性である、請求項1記載の方法。
- nが3又は4であり、Bがプリン、デアザプリン又はピリミジン塩基である、請求項1乃至請求項8のいずれか1項記載の方法。
- 前記糖部分が、リボシル、2′−デオキシリボシル、3′−デオキシリボシル、2′,3′−ジデオキシリボシル、2′,3′−ジデヒドロジデオキシリボシル、2′−アルコキシリボシル、2′−アジドリボシル、2′−アミノリボシル、2′−フルオロリボシル、2′−メルカプトリボシル、2′−アルキルチオリボシル、3′−アルコキシリボシル、3′−アジドリボシル、3′−アミノリボシル、3′−フルオロリボシル、3′−メルカプトリボシル、3′−アルキルチオリボシル、炭素環式、非環式及び他の修飾糖からなる群から選択される、請求項1乃至請求項9のいずれか1項記載の方法。
- 前記塩基が、ウラシル、チミン、シトシン、グアニン、7−デアザグアニン、ヒポキサンチン、7−デアザヒポキサンチン、アデニン、7−デアザアデニン、2,6−ジアミノプリン及びそのアナログからなる群から選択される、請求項1乃至請求項10のいずれか1項記載の方法。
- 前記酵素活性化可能標識が、5−ブロモ−4−クロロ−3−インドリルリン酸、3−インドキシルリン酸及びp−ニトロフェニルリン酸からなる群から選択される発色色素である、請求項1記載の方法。
- 前記末端リン酸標識ヌクレオチドにおけるP−Lが、2−(5′−クロロ−2′−ホスホリルオキシフェニル)−6−クロロ−4−(3H)−キナゾリノン、フルオレセイン二リン酸、フルオレセイン3′(6′)−O−アルキル−6′(3′)−リン酸、9H−(1,3−ジクロロ−9,9−ジメチルアクリジン−2−オン−7−イル)リン酸、4−メチルウンベリフェリルリン酸、レゾルフィンリン酸、4−トリフルオロメチルウンベリフェリルリン酸、ウンベリフェリルリン酸、3−シアノウンベリフェリルリン酸、9,9−ジメチルアクリジン−2−オン−7−イルリン酸及び6,8−ジフルオロ−4−メチルウンベリフェリルリン酸からなる群から選択される蛍光発生色素を含むリン酸化標識である、請求項1記載の方法。
- 前記化学発光化合物がアルカリホスファターゼ活性化1,2−ジオキセタン化合物である、請求項1記載の方法。
- 前記1,2−ジオキセタン化合物が2−クロロ−5−(4−メトキシスピロ[1,2−ジオキセタン−3,2′−(5−クロロ−)トリシクロ[3,3,1−13,7]−デカン]−1−イル)−1−フェニルリン酸、クロロアダマンタ−2′−イリデンメトキシフェノキシリン酸化ジオキセタン及び3−(2′−スピロアダマンタン)−4−メトキシ−4−(3″−ホスホリルオキシ)フェニル−1,2−ジオキセタンからなる群から選択される、請求項14記載の方法。
- 前記核酸ポリメラーゼ反応が複数サイクル実施される、請求項1乃至請求項15のいずれか1項記載の方法。
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PCT/US2003/027286 WO2004020604A2 (en) | 2002-08-29 | 2003-08-29 | Allele specific primer extension |
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US8361712B2 (en) * | 2007-12-17 | 2013-01-29 | General Electric Company | Contamination-free reagents for nucleic acid amplification |
US8507662B2 (en) | 2001-01-19 | 2013-08-13 | General Electric Company | Methods and kits for reducing non-specific nucleic acid amplification |
US7223541B2 (en) * | 2001-08-29 | 2007-05-29 | Ge Healthcare Bio-Sciences Corp. | Terminal-phosphate-labeled nucleotides and methods of use |
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US5270185A (en) * | 1989-04-21 | 1993-12-14 | Hoffmann-La Roche Inc. | High-efficiency cloning of CDNA |
US5518900A (en) * | 1993-01-15 | 1996-05-21 | Molecular Tool, Inc. | Method for generating single-stranded DNA molecules |
US5759772A (en) * | 1992-07-23 | 1998-06-02 | Wisconsin Alumni Research Foundation | Method for determining the sex of an embryo |
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ATE319857T1 (de) * | 1998-12-14 | 2006-03-15 | Li Cor Inc | Kit und methode zur nukleinsäuresequenzierung einzelner moleküle durch polymerase synthese |
US7056661B2 (en) * | 1999-05-19 | 2006-06-06 | Cornell Research Foundation, Inc. | Method for sequencing nucleic acid molecules |
US6399335B1 (en) * | 1999-11-16 | 2002-06-04 | Advanced Research And Technology Institute, Inc. | γ-phosphoester nucleoside triphosphates |
AU2001268291A1 (en) * | 2000-06-07 | 2001-12-17 | Li-Cor, Inc. | Charge-switch nucleotides |
JP3638516B2 (ja) * | 2000-09-28 | 2005-04-13 | 株式会社日立製作所 | 核酸検出方法および核酸検出キット |
AU2002227156A1 (en) * | 2000-12-01 | 2002-06-11 | Visigen Biotechnologies, Inc. | Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity |
US7052839B2 (en) * | 2001-08-29 | 2006-05-30 | Amersham Biosciences Corp | Terminal-phosphate-labeled nucleotides and methods of use |
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US7033762B2 (en) * | 2001-08-29 | 2006-04-25 | Amersham Biosciences Corp | Single nucleotide amplification and detection by polymerase |
US6546720B2 (en) * | 2001-09-04 | 2003-04-15 | Ford Global Technologies, Inc. | Method and apparatus for controlling the amount of reactant to be added to a substance using a sensor which is responsive to both the reactant and the substance |
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