| (11) | EP 3 690 039 B1 |
(12) | EUROPEAN PATENT SPECIFICATION |
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(54) | METHOD FOR DETECTING SINGLE BASE SUBSTITUTION USING ION EXCHANGE CHROMATOGRAPHY VERFAHREN ZUM NACHWEIS VON EINZELBASENSUBSTITUTION MITTELS IONENAUSTAUSCHCHROMATOGRAFIE PROCÉDÉ DE DÉTECTION D'UNE SUBSTITUTION DE BASE UNIQUE À L'AIDE DE LA CHROMATOGRAPHIE PAR ÉCHANGE D'IONS |
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Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). |
TECHNICAL FIELD
BACKGROUND ART
CITATION LIST
PATENT LITERATURE
NON PATENT LITERATURE
SUMMARY OF INVENTION
TECHNICAL PROBLEM
SOLUTION TO PROBLEM
the sequence of the first primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having a first allele at the polymorphic site, and any one or two or three out of three bases at the 3' end or one or both of two bases at the 3' end of the sequence of the first primer corresponds to the polymorphism site, wherein
the sequence of the second primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having a second allele at the polymorphic site, and any one or two or three out of three bases at the 3' end or one or both of two bases at the 3' end of the sequence of the second primer corresponds to the polymorphism site, wherein
the sequence of the third primer does not include the polymorphic site and is complementary to the first strand of the double-stranded deoxyribonucleic acid, wherein
a non-nucleotide component is added to at least one of the first primer and the second primer, wherein
the non-nucleotide component is preferably an ionic functional group selected from the group consisting of a hydroxy group, an aldehyde group, a carboxy group, an amino group, a nitro group, a nitroso group, a thiol group, a sulfonic acid group, a fluoro group, a chloro group, a bromo group, and iodine group, or a molecule containing at least one or more of the ionic functional groups, more preferably a fluorescent substance described in Table 1;
the non-nucleotide component is preferably an ionic functional group selected from the group consisting of a hydroxy group, an aldehyde group, a carboxy group, an amino group, a nitro group, a nitroso group, a thiol group, a sulfonic acid group, a fluoro group, a chloro group, a bromo group, and iodine group, or a molecule containing at least one or more of the ionic functional groups, more preferably a fluorescent substance described in Table 1.
ADVANTAGEOUS EFFECTS OF INVENTION
DESCRIPTION OF EMBODIMENTS
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
5-FAM | 5-Carboxyfluorescein | 3' ,6'-dihydroxy-3-oxospiro[2-benzofuran-1 ,9'- xanthene]-5-carboxylic acid | C1=CC2=C(C=C1C(=O)O)C(=O)OC23C4=C (C=C(C=C4)O)OC5=C3C=CC(=C5)O |
5-ROX | 5-carboxy-X-rhodamine | 16-(2-carboxy-4-carboxylatophenyl)-3-oxa-9 λ5,23-diazaheptacyclo[17.7.1.15,9.02,17.04,15.0 23,27.013,28]octacosa-1,4,9(28),13,15,17,19(27)-heptaen-9-ylium | C1CC2=C3C(=C4C(=C2)C(=C5C=C6CCC[N +]7=C6C(=C5O4)CCC7)C8=C(C=C(C=C8)C (=O)[O-])C(=O)O)CCCN3C1 |
6-FAM | 6-Carboxyfluorescein | 3',6'-dihydroxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylic acid | C1=CC2=C(C=C1C(=O)O)C3(C4=C(C=C(C =C4)O)OC5=C3C=CC(=C5)O)OC2=O |
6-JOE | 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein | 4',5'-dichloro-3',6'-dihydroxy-2',7'-dimethoxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylic acid | COC1=C(C(=C2C(=C1)C3(C4=CC(=C(C(=C 4O2)Cl)O)OC)C5=C(C=CC(=C5)C(=O)O)C( =O)O3)Cl)O |
6-ROX | 6-Carboxy-X-rhodamine | 16-(2-carboxy-5-carboxylatophenyl)-3-oxa-9 λ5,23-diazaheptacyclo[17.7.1.15,9.02,17.04,15.0 23,27.013,28]octacosa-1,4,9(28),13,15,17,19(27)-heptaen-9-ylium | C1CC2=C3C(=C4C(=C2)C(=C5C=C6CCC[N +]7=C6C(=C5O4)CCC7)C8=C(C=CC(=C8)C (=O)[O-])C(=O)O)CCCN3C1 |
6-TET | 6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimiyl ester | (2,5-dioxopyrrolidin-1-yl) 2',4,7,7'-tetrachloro-3',6'-dihydroxy-1-oxospiro[2-benzofuran-3,9'-xanthene]-5-carboxylate | C1CC(=O)N(C1=O)OC(=O)C2=CC(=C3C(= C2Cl)C4(C5=CC(=C(C=C5OC6=CC(=C(C= C64)Cl)O)O)Cl)OC3=O)Cl |
\Product name | Synonym | IUPAC name | Canonical SMILES/Isometric SMILES |
Alexa Fluor 350 | 7-amino-4-methyl-6-sulfocoumarin-3-acetic acid | 7-amino-3-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxoethyl]-4-methyl-2-oxochromene-6- sulfonic acid | CC1=C(C(=O)OC2=CC(=C(C=C12)S(=O)(= O)O)N)CC(=O)ON3C(=O)CCC3=O |
Alexa Fluor 430(1-) | N/A | [9-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-8,8-dimethyl-2-oxo-4-(trifluoromethyl)pyrano[3,2-g]quinolin-6-yl]methanesulfonate | CC1 (C=C(C2=C(N1CCCCCC(=O)ON3C(=O )CCC3=O)C=C4C(=C2)C(=CC(=O)O4)C(F)( F)F)CS(=O)(=O)[O-])C |
Alexa Fluor 480(3-) | N/A | 3-(3-amino-6-imino-4,5-disulfonatoxanthen-9-yl)-4-carboxybenzoate | C1=CC(=C(C=C1C(=O)[O-])C2=C3C=CC(=N)C(=C3OC4=C2C=CC(=C 4S(=O)(=O)[O-])N)S(=O)(=O)[O-])C(=O)O |
Alexa Fluor 488 meta-isomer | dilithium 5-carboxy-2-(3,6-diamino-4,5-disulfonatoxanthenium-9-yl)benzoate | dilithium ;3-amino-9-(2,4-dicarboxyphenyl)-6-iminoxanthene-4,5-disulfonate | [Li+].[Li+].C1=CC(=C(C=C1C(=O)O)C(=O)O )C2=C3C=CC(=N)C(=C3CC4=C2C=CC(=C4 S(=O)(=O)[O-])N)S(=O)(=O)[O-] |
Alexa Fluor 488 meta-isomer(2-) | 5-carboxy-2-(3,6-diamino-4,5-disulfonatoxanthenium-9-yl)benzoate | 3-amino-9-(2,4-dicarboxyphenyl)-6-iminoxanthene-4,5-disulfonate | C1=CC(=C(C=C1C(=O)O)C(=O)O)C2=C3C =CC(=N)C(=C3OC4=C2C=CC(=C4S(=O)(= O)[O-])N)S(=O)(=O)[O-] |
Alexa Fluor 488 para-isomer | dilithium 4-carboxy-2-(3,6-diamino-4,5-disulfonatoxanthenium-9-yl)benzoate | dilithium;3-amino-9-(2,5-dicarboxyphenyl)-6-iminoxanthene-4,5-disulfonate | [Li+].[Li+].C1=CC(=C(C=C1C(=O)O)C2=C3 C=CC(=N)C(=C3OC4=C2C=CC(=C4S(=O)( =O)[O-])N)S(=O)(=O)[O-])C(=O)O |
Product name | Synonym | IUPAC name | Canonical SMILES/Isometric SMILES |
Alexa Fluor 488 para-isomer(2-) | 4-carboxy-2-(3,6-diamino-4,5-disulfonatoxanthenium-9-yl)benzoate | 3-amino-9-(2,5-dicarboxyphenyl)-6-iminoxanthene-4,5-disulfonate | C1=CC(=C(C=C1C(=O)O)C2=C3C=CC(=N) C(=C3OC4=C2C=CC(=C4S(=O)(=O)[O-])N)S(=O)(=O)[O-])C(=O)O |
Alexa Fluor 514 meta-isomer | 6-(2-carboxy-4-{[(2.5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-9-iminio-2,2,4-trimethyl-12-sulfo-1,3,4,9-tetrahydro-2H-chromeno[3,2-g]quinoline-10-sulfonate | 9-amino-6-[2-carboxy-4-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]-2,2,4-trimethyl-12-sulfo-3,4-dihydrochromeno[3,2-g]quinolin-1-ium-10-sulfonate | CC1CC([NH+]=C2C1=CC3=C(C4=C(C(=C( C=C4)N)S(=O)(=O)[O-])OC3=C2S(=O)(=O)O)C5=C(C=C(C=C5)C( =O)ON6C(=O)CCC6=O)C(=O)O)(C)C |
Alexa Fluor 514 para-isomer | 6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-9-iminio-2,2,4-trimethyl-12-sulfo-1,3,4,9-tetrahydro-2H-chromeno[3,2-g]quinoline-10-sulfonate | 9-amino-6-[2-carboxy-5-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]-2,2,4-trimethyl-12-sulfo-3,4-dihydrochromeno[3,2-g]quinolin-1-ium-10-sulfonate | CC1CC([NH+]=C2C1=CC3=C(C4=C(C(=C( C=C4)N)S(=O)(=O)[O-])OC3=C2S(=O)(=O)O)C5=C(C=CC(=C5)C( =O)ON6C(=O)CCC6=O)C(=O)O)(C)C |
Alexa Fluor 532 | N/A | 12-(4-carboxyphenyl)-7,8,8,16,16,17-hexamethyl-4,20-disulfo-2-oxa-6,18-diazapentacyclo[11.7.0.03,11.05,9.015,19]icosa-1(20),3,5,9,11,13,15(19)-heptaen-6-ium | CC1C(C2=C(N1)C(=C3C(=C2)C(=C4C=C5C (=[NH+]C(C5(C)C)C)C(=C403)S(=O)(=O)O) C6=CC=C(C=C6)C(=O)O)S(=O)(=O)O)(C)C |
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
Alexa Fluor 546 | N/A | (2-{[4-carboxy-2,3,6-trichloro-5-(2,2,4,8,10,10-hexamethyl-12,14-disulfo-2,3,4,8,9,10-hexahydro-1H-13-oxa-1,11-diazapentacen-6-yl)phenyl]sulfanyl}-1-hydroxyethylidene)({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl})azanium | CC1CC(NC2=C1C=C3C(=C4C=C5C(CC(N= C5C(=C4OC3=C2S(=O)(=O)O)S(=O)(=O)O) (C)C)C)C6=C(C(=C(C(=C6Cl)SCC(=[NH+]C CCCCC(=O)ON7C(=O)CCC7=O)O)Cl)Cl)C( =O)O)(C)C |
Alexa Fluor 555 | N/A | 4-(3-amino-6-imino-4,5-disulfoxanthen-9-yl)benzene-1,3-dicarboxylic acid | C1=CC(=C(C=C1C(=O)O)C(=O)O)C2=C3C =CC(=N)C(=C3OC4=C2C=CC(=C4S(=O)(= O)O)N)S(=O)(=O)O |
Alexa Fluor 568 ortho-isomer | [6-(2-carboxy-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-2,2,10,10-tetramethyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g']diquinolin-1-ium-4-yl]methanesulfonate | 6-(2-carboxy-4-{[(2,5-d)oxopyrro)id)n-1-yl)oxy]carbonyl}phenyl)-2,2,10,10-tetramethyl-4-(sulfomethyl)-8-(sulfonatomethyl)-2,10-dihydro-1H-13-oxa-1,11-diazapentacen-11-ium | CC1(C=C(C2=CC3=C(C=C2N1)OC4=CC5=[ NH+]C(C=C(C5=CC4=C3C6=C(C=C(C=C6) C(=O)ON7C(=O)CCC7=O)C(=O)O)CS(=O)( =O)[O-])(C)C)CS(=O)(=O)O)C |
Alexa Fluor 568 para-isomer | [6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-2,2,10,10-tetramethyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g']diquinolin-1-ium-4-yl]methanesulfonate | 6-(2-carboxy-5-{[(2.5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-2,2,10, 10-tetramethyl-4-(sulfomethyl)-8-(sulfonatomethyl)-2,10-dihydro-1H-13-oxa-1,11-diazapentacen-11-ium | CC1(C=C(C2=CC3=C(C=C2N1)OC4=CC5=[ NH+]C(C=C(C5=CC4=C3C6=C(C=CC(=C6) C(=O)ON7C(=O)CCC7=O)C(=O)O)CS(=O)( =O)[O-])(C)C)CS(=O)(=O)O)C |
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
Alexa Fluor 594 meta-isomer | [6-(2-carboxy-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1,2,2,10,10,11-hexamethyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g']diquinolin-1-ium-4-yl]methanesulfonate | 6-(2-carboxy-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1,2,2,10,10,11- hexamethyl-4-(sulfomethyl)-8- (sulfonatomethyl)-2,10-dihydro-1H-13-oxa- 1,11-diazapentacen-11-ium | CC1(C=C(C2=CC3=C(C=C2N1C)OC4=CC5 =[N+](C(C=C(C5=CC4=C3C6=C(C=C(C=C6 )C(=O)ON7C(=O)CCC7=O)C(=O)O)CS(=O)( =O)[O-])(C)C)C)CS(=O)(=O)O)C |
Alexa Fluor 594 para-isomer | [6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1,2,2,10,10,11-hexamethyl-8-(sulfomethyl)-10,11-dihydro-2H-pyrano[3.2-g:5,6-g'}diquinolin-1-ium-4-yl]methanesulfonate | 6-(2-carboxy-5-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}phenyl)-1,2,2,10,10,11-hexamethyl-4-(sulfomethyl)-8-(sulfonatomethyl)-2,10-dihydro-1H-13-oxa-1,11-diazapentacen-11-ium | CC1(C=C(C2=CC3=C(C=C2N1C)OC4=CC5 =[N+](C(C=C(C5=CC4=C3C6=C(C=CC(=C6 )C(=O)ON7C(=O)CCC7=O)C(=O)O)CS(=O)( =O)[O-])(C)C)C)CS(=O)(=O)O)C |
Alexa Fluor 610-X | bis(N, N-diethylethanaminium) 2,3,5-trichloro-4-{[2-({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]sulfanyl}-6-[1.2,2,10,10,11-hexamethyl-4,8-bis(sulfonatomethyl)-10,11-dihydro-2H-pyrano[3,2-g:5,6-g']diquinolin-1-ium-6-yl]benzoate | 6-(2-carboxylato-3,4,6-trichloro-5-{[({6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}carbamoyl)methyl]sulfanyl}phenyl)-1,2,2,10,10,11-hexamethyl-4,8-bis(suffonatomethyl)-2,10-dihydro-1H-13-oxa-1,11-diazapentacen-11-ium; bis(triethylazanium) | CC[NH+](CC)CC.CC[NH+](CC)CC.CC1(C= C(C2=CC3=C(C=C2N1C)OC4=CC5=[N+](C( C=C(C5=CC4=C3C6=C(C(=C(C(=C6Cl)SCC (=O)NCCCCCC(=O)ON7C(=O)CCC7=O)Cl) Cl)C(=O)[O-])CS(=O)(=O)[-O ])(C)C)C)CS(=O)(=O)[O-])C |
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
Alexa Fluor 647 | N/A | 2-[5-[3,3-dimethyl-5-sulfo-1-(3- sulfopropyl)indol-1-ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6- phosphonooxyhexylamino)pentyl]-1-(3- sulfopropyl)indole-5-sulfonic acid | CC1(C2=C(C=CC(=C2)S(=O)(=O)O)[N+](=C 1C=CC=CC=C3C(C4=C(N3CCCS(=O)(=O) O)C=CC(=C4)S(=O)(=O)O)(C)CCCCC(=O) NCCCCCCO[P+](O)(O)[O-])CCCS(=O)(=O)O)C |
Atto 425 | 4-[3-(ethoxycarbonyl)-6,8,8-trimethyl-2-oxo-7,8-dihydro-2H-pyrano[3,2-g]quinolin-9(6H)-yl]bulanoic acid | 4-(3-ethoxycarbonyl-6,8,8-trimethyl-2-oxo-6,7-dihydropyrano[3,2-g]quinolin-9-yl)butanoic acid | CCOC(=O)C1=CC2=CC3=C(C=C2OC1=O) N(C(CC3C)(C)C)CCCC(=O)O |
Atto 465 | N/A | 4-(3-amino-6-iminoacridin-10-yl)butanoic acid;perchloric acid | C1=CC(=N)C=C2C1=CC3=C(N2CCCC(=O) O)C=C(C=C3)N.OCl(=O)(=O)=O |
Atto 488 | 10-(3-carboxypropyl)-3,6-bis(dimethylamino)acridinium perchlorate | 4-[3,6-bis(dimethylamino)acridin-10-ium-10-yl]butanoic acid;perchlorate | CN(C)C1=CC2=C(C=C1)C=C3C=CC(=CC3 =[N+]2CCCC(=O)O)N(C)C.[O-]Cl(=O)(=O)=O |
Atto 520 | N-[9-(2-carboxyethyl)-6-(ethylamino)-2,7-dimethyl-3H-xanthen-3-ylidene]ethanaminium perchlorate | [9-(2-carboxyethyl)-6-(ethylamino)-2,7-dimethylxanthen-3-ylidene]-ethylazanium;perchlorate | CCNC1=C(C=C2C(=C1)OC3=CC(=[NH+]CC )C(=CC3=C2CCC(=O)O)C)C.[O-]Cl(=O)(=O)=O |
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
Atto 532 | N/A | 4-[[2-[3-(ethylamino)-6-ethylimino-4,5-disulfoxanthen-9-yl]benzoyl]-methylamino]butanoic acid | CCNC1=C(C2=C(C=C1)C(=C3C=CC(=NCC) C(=C3O2)S(=O)(=O)O)C4=CC=CC=C4C(= O)N(C)CCCC(=O)O)S(=O)(=O)O |
Atto 610 | 1-(3-carboxypropyl)-9-(dimethylamino)-11,11-dimethyl-2,3,4,11-tetrahydronaphtho[2,3-g]quinolinium perchlorate | 4-[9-(dimethylamino)-11,11-dimethyl-3,4-dihydro-1-ium-1-yl]butanoic acid;perchlorate | CC1 (C2=CC3=[N+](CCCC3=CC2=CC4=C1 C=C(C=C4)N(C)C)CCCC(=O)O)C.[O-]Cl(=O)(=O)=O |
Atto 635 | 1-(3-carboxypropyl)-9-(dimethylamino)-2,2,4,11,11-pentamethyl-2,11-dihydronaphtho[2,3-g]quinolinium perchlorate | 4-(9-(dimethylamino)-2.2.4.11,11-pentamethylnaphtho[2,3-g]quinolin-1-ium-1-yl]butanoic acid;perchlorate | CC1=CC([N+](=C2C1=CC3=CC4=C(C=C(C =C4)N(C)C)C(C3=C2)(C)C)CCCC(=O)O)(C) C.[O-]Cl(=O)(=O)=O |
Atto 655 | N/A | 1-(3-carboxypropyl)-11-ethyl-2,2-dimethyl-4-(sulfonatomethyl)-2,3,4,8,9,10-hexahydro-1H-13-oxa-1,6.11-triazapentacen-11-ium | CC[N+]1=C2C=C3C(=NC4=C(O3)C=C5C(= C4)C(CC(N5CCCC(=O)O)(C)C)CS(=O)(=O)[ O-])C=C2CCC1 |
Cy3 | 2-((1E,3E)-3-(1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)prop-1-en-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium-5-sulfonate | (2Z)-2-[(E)-3-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindol-1-ium-2-yl]prop-2-enylidene]-1-ethyl-3,3-dimethylindole-5-sulfonate | CCN\1C2=C(C=C(C=C2)S(=C)(=O)[O-])C(/C1=C/C=C/C3=[N+](C4=C(C3(C)C)C=C (C=C4)S(=O)(=O)O)CCCCCC(=O)O)(C)C |
Product name | Synonym | IUPAC name | Canonical SMILES/Isomeric SMILES |
Cy3.5 | Cy3.5 carboxylic acid | 6-[(2E)-1,1-dimethyl-2-[(E)-3-(1,1,3- trimethylbenzo[e]indol-3-ium-2-yl)prop-2- enylidene]benzo[e]indol-3-yl]hexanoic acid;chloride | CC1(C(=[N+](C2=C1C3=CC=CC=C3C=C2) C)/C=C/C=C/4\C(C5=C(N4CCCCCC(=O)O) C=CC6=CC=CC=C65)(C)C)C.[Cl-] |
Cy5 | 2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium-5-sulfonate | (2Z)-2-[(2E,4E)-5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindol-1-ium-2-yl]penta-2,4-dienylidene]-1-ethyl-3,3-dimethylindole-5-sulfonate | CCN\1C2=C(C=C(C=C2)S(=O)(=O)[O-])C(/C1=C/C=C/C=C/C3=[N+](C4=C(C3(C)C )C=C(C=C4)S(=O)(=O)O)CCCCCC(=O)O)( C)C |
Cy5.5 | N/A | (2Z)-2-[(2E,4E)-5-[3-(5-carboxypenty))-1.1-dimethyl-6,8-disulfobenzo[e]indol-3-ium-2-yl]penta-2,4-dienylidene]-3-ethyl-1,1-dimethyl-8-(trioxidanylsulfanyl)benzo[e]indole-6-sulfonate | CCN\1C2=C(C3=CC(=CC(=C3C=C2)S(=O)( =O)[O-])SOOO)C(/C1=C/C=C/C=C/C4=[N+](C5=C( C4(C)C)C6=CC(=CC(=C6C=C5)S(=O)(=O) O)S(=O)(=O)O)CCCCCC(=O)O)(C)C |
Digoxigenin | Lanadigenin | 3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12, 14-trihydroxy-1 0, 13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]-2H-furan-5-one | C[C@]12CC[C@@H](C[C@H]1CC[C@@H] 3[C@@H]2C[C@H]([C@]4([C@@]3(CC[C @@H]4C5=CC(=O)OC5)O)C)O)O |
FITC | Fluorescein 5 isothiocyanate | 3',6'-dihydroxy-6-isothiocyanatospiro[2-benzofuran-3,9'-xanthene]-1 -one | C1=CC2=C(C=C1N=C=S)C(=O)OC23C4=C( C=C(C=C4)O)OC5=C3C=CC(=C5)O |
Product name | Synonym | IUPAC name | |
TAMRA | Tetramethylrhodamine | 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9- yl]benzoate | CN(C)C1=CC2=C(C=C1)C(=C3C=CC(=[N+] (C)C)C=C3O2)C4=CC=CC=C4C(=O)[O-] |
Texas Red | sulforhodamine sulfonyl chloride | 16-[4-(chlorosulfonyl)-2-sulfonatophenyl]-3-oxa-9λ5,23-diazaheptacyclo[17.7.1.15,902,1 7.04,15.023,27.013,28]octacosa-1,4,9(28),13,15,17,19(27)-heptaen-9-ylium | C1CC2=C3C(=C4C(=C2)C(=C5C=C6CCC[N +]7=C6C(=C5O4)CCC7)C8=C(C=C(C=C8)S (=O)(=O)Cl)S(=O)(=O)[O-])CCCN3Cl |
BRIEF DESCRIPTION OF DRAWINGS
[Fig. 1] Fig. 1 shows a result of overlaying elution peaks of amplification products according to three fluorescently-labelled primers (SEQ ID NOs: 3, 7, and 8).
[Fig. 2] Fig. 2 shows separation and detection of amplification products from ∗6 polymorphic sites of the UGT1A1 gene using non-nucleotide component-added ASPs.
[Fig. 3] Fig. 3 shows separation and detection of amplification products from a periphery of the codon 515 site of the MPL gene using non-nucleotide component-added ASPs.
EXAMPLES
[Example 1] Amplification Product from ∗6 Polymorphic Site of UGT1A1 Gene Using Non-Nucleotide Component-Added ASPs
SEQ ID | 5' to 3' primer sequences | Bases (bp) | Non-nucleotide components | Excitation wavelength (nm) | Fluorescence wavelength (nm) |
(Forward primers) | |||||
1 | GTTGTACATCAGAGACATA | 19 | Unlabeled | - | - |
2 | Alexa488-GTTGTACATCAGAGACATA | 19 | Alexa488 | 490 | 519 |
3 | FAM-GTTGTACATCAGAGACATA | 19 | FAM | 495 | 520 |
4 | ATTO488-GTTGTACATCAGAGACATA | 19 | ATTO488 | 502 | 522 |
5 | Cy3-GTTGTACATCAGAGACATA | 19 | Cy3 | 552 | 570 |
6 | Alexa546-GTTGTACATCAGAGACATA | 19 | Alexa546 | 556 | 573 |
7 | TAMRA-GTTGTACATCAGAGACATA | 19 | TAMRA | 565 | 580 |
8 | Cy3.5-GTTGTACATCAGAGACATA | 19 | Cy3.5 | 581 | 596 |
9 | Cy5-GTTGTACATCAGAGACATA | 19 | Cy5 | 643 | 667 |
10 | Cy5.5-GTTGTACATCAGAGACATA | 19 | Cy5.5 | 675 | 694 |
11 | DIG-GTTGTACATCAGAGACATA | 19 | DIG | - | - |
(Common reverse primer) | |||||
12 | GAATCATTCTCAAAAACATTATGCCC | 19 | Unlabeled | - | - |
Reagents, Amplification Conditions, and Ion-Exchange Chromatography Conditions
[Reagents] | |
5×buffer (for Q5) | 5µL |
10mM dNTP | 0.5µL |
each of 10 µM forward primers | 1.25µL |
10 µM reverse primer | 1.25µL |
2000 U/mL Q5 DNA polymerase | 0.25µL |
Nuclease-free Water | 11.75µL |
DNA specimen (25 ng) | 5µL |
[Amplification Conditions] | |
98°C for 30 seconds | |
98°C for 10 seconds, 58°C for 20 seconds (40 cycles) |
[Ion-Exchange Chromatography Conditions] | |
HPLC anion ion-exchange resin column: TSKgelDNA-NPR (TOSOH CORPORATION) | |
Eluent: 20 mM Tris-HCl(pH 9.0), 0.5-0.7 M NaCl gradient (10 min) | |
Flow rate: 0.75 mL/min | |
Column oven: 25°C | |
Detector: UV wavelength 260 nm (even non-fluorescent substances are detectable at the selected UV wavelength) |
Elution time (min) | Δ (min) | |
ATTO488 | 8.51 | -0.22 |
Cy3 | 8.53 | -0.19 |
TAMRA | 8.54 | -0.18 |
DIG | 8.63 | -0.09 |
Cy5 | 8.64 | -0.08 |
Unlabeled | 8.73 | - |
Alexa488 | 8.83 | 0.10 |
Alexa546 | 8.93 | 0.20 |
Cy3.5 | 9.07 | 0.34 |
FAM | 9.28 | 0.55 |
Cy5.5 | 9.50 | 0.78 |
[Example 2] Separation and Detection of Amplification Products from ∗6 Polymorphic Site of UGT1A1 Gene Using Non-Nucleotide Component-Added ASPs
Reagents, Amplification Conditions, and Ion-Exchange Chromatography Conditions
[Reagents] | |
5× buffer (for Q5) | 5µL |
10 mM dNTP | 0.5µL |
10 µM forward primer (SEQ ID NO: 3) | 1.25µL |
10 µM reverse primer (SEQ ID NO: 13) | 1.25µL |
10 µM reverse primer | 1.25µL |
2000 U/mL Q5 DNA polymerase | 0.25µL |
Nuclease-free Water | 10.5µL |
DNA specimen (25 ng) | 5µL |
[Example 3] Separation and Detection of MPL Gene Mutations (Codon 515) Using Non-Nucleotide Component-Added ASPs
SEQ ID NO: 14 (ASP for W515L)
5'-CTGCTGCTGCTGAGGTTTC-3'
SEQ ID NO: 15 (ASP for W515K)
5'-CTGCTGCTGCTGAGGAA-3'
SEQ ID NO: 16 (ASP for W515A)
5'-TGCTGCTGCTGAGCGC-3'
SEQ ID NO: 17 (common reverse primer)
5'-GGCGGTACCTGTAGTGTGC-3'
SEQ ID NO: 18 (ASP for biotin-labeled W515K)
5'-Biotin-CTGCTGCTGCTGAGGAA-3'
SEQ ID NO: 19 (ASP for amino-group-labeled W515K)
5'-NH2-CTGCTGCTGCTGAGGAA-3'
SEQ ID NO: 20 (ASP for Cy3.5 fluorescent-dye-labeled W515K)
5'-Cy3.5-CTGCTGCTGCTGAGGAA-3'
SEQ ID NO: 21 (W515L gene mutant sequence)
[Chem 1]
SEQ ID NO: 22 (W515K gene mutant sequence)
[Chem 2]
SEQ ID NO: 23 (W515A gene mutant sequence)
[Chem 3]
Reagents, Amplification Conditions, and Ion-Exchange Chromatography Conditions
[Reagents] | |
5× buffer (for Q5) | 5µL |
10 mM dNTP | 0.5µL |
10 µM forward primer (SEQ ID NO: 14) | 1.25µL |
10 µM forward primer | |
(any of SEQ ID NOs: 15 and 18 to 20) | 0.31µL |
10 µM forward primer (SEQ ID NO: 16) | 0.25µL |
10 µM reverse primer (SEQ ID NO: 17) | 1.25µL |
20×EvaGreen | 1.25µL |
2000 U/mL Q5 DNA polymerase | 0.25µL |
Nuclease-free Water | 9.94µL |
DNA specimen | 5µL |
(1500 copies of linear plasmid DNA [SEQ ID NOs: 21 to 23] cleaved by an appropriate restriction enzyme) |
[Amplification Conditions] | |
98°C: 30 seconds | |
98°C: 10 seconds, 62°C: 20 seconds (35 cycles) |
[Ion-Exchange Chromatography Conditions] | |
HPLC anion ion-exchange resin column: TSKgelDNA-NPR (TOSOH CORPORATION) | |
Eluent: 20mM Tris-HCl(pH9.0), 0.47-0.62M NaCl gradient (10min) | |
Flow rate: 0.75mL/min | |
Column oven: 25°C | |
Detector: UV wavelength 260 nm (even non-fluorescent substances are detectable at the selected UV wavelength) |
INDUSTRIAL APPLICABILITY
SEQUENCE LISTING
<110> SEKISUI MEDICAL co. LTD.
<120> Sigle nucleotide substitution detection method using ion exchange chromatography
<130> 17P01204
<160> 23
<170> PatentIn version 3.5
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(a) providing a sample containing a double-stranded deoxyribonucleic acid containing a polymorphic site;
(b) providing a first primer, a second primer, and a third primer, wherein
the sequence of the first primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having a first allele at the polymorphic site, and any one or two or three out of three bases at the 3' end or one or both of two bases at the 3' end of the sequence of the first primer corresponds to the polymorphism site, wherein
the sequence of the second primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having a second allele at the polymorphic site, and any one or two or three out of three bases at the 3' end or one or both of two bases at the 3' end of the sequence of the second primer corresponds to the polymorphism site, wherein
the sequence of the third primer does not include the polymorphic site and is complementary to the first strand of the double-stranded deoxyribonucleic acid, wherein
a non-nucleotide component is added to at least one of the first primer and the second primer;
(c) performing a polymerase chain reaction, wherein
the polymerase chain reaction is performed under a condition that strand elongation due to a polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the first allele preferentially occurs as compared to strand elongation due to a polymerase from the second primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the first allele, and that strand elongation due to a polymerase from the second primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the second allele preferentially occurs as compared to strand elongation due to a polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the second allele;
(d) subjecting amplification products of the polymerase chain reaction to ion-exchange chromatography, wherein
the difference in size of the amplification product of the polymerase chain reaction from the first primer and the third primer and the amplification product of the polymerase chain reaction from the second primer and the third primer is 0 base pair, 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or 10 base pairs; and
(e) detecting the presence of one or both of the first and second alleles based on elution positions or elution times of the amplification products,
wherein the non-nucleotide component is a substance inducing a change in electric charge at the 5' end of the primer.
(a) Bereitstellen einer Probe, die eine eine polymorphe Stelle enthaltende doppelsträngige Desoxyribonukleinsäure enthält;
(b) Bereitstellen eines ersten Primers, eines zweiten Primers und eines dritten Primers, wobei
die Sequenz des ersten Primers zu dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit einem ersten Allel an der polymorphen Stelle komplementär ist, und irgendeine oder zwei oder drei von drei Basen an dem 3'-Ende oder eine oder beide von zwei Basen an dem 3'-Ende der Sequenz des ersten Primers der polymorphen Stelle entspricht, wobei
die Sequenz des zweiten Primers zu dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit einem zweiten Allel an der polymorphen Stelle komplementär ist, und irgendeine oder zwei oder drei von drei Basen an dem 3'-Ende oder eine oder beide von zwei Basen an dem 3'-Ende der Sequenz des zweiten Primers der polymorphen Stelle entspricht, wobei
die Sequenz des dritten Primers nicht die polymorphe Stelle einschließt und komplementär zu dem ersten Strang der doppelsträngigen Desoxyribonukleinsäure ist, wobei
ein nicht-Nukleotid-Bestandteil mindestens einem von dem ersten Primer und dem zweiten Primer hinzugefügt wird;
(c) Durchführen einer Polymerasekettenreaktion, wobei
die Polymerasekettenreaktion unter einer Bedingung durchgeführt wird, dass Strangverlängerung auf Grund einer Polymerase von dem ersten Primer, der mit dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit dem ersten Allel hybridisiert ist, bevorzugt stattfindet, verglichen mit Strangverlängerung auf Grund einer Polymerase von dem zweiten Primer, der mit dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit dem ersten Allel hybridisiert ist, und, dass Strangverlängerung auf Grund einer Polymerase von dem zweiten Primer, der mit dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit dem zweiten Allel hybridisiert ist, bevorzugt stattfindet, verglichen mit Strangverlängerung auf Grund einer Polymerase von dem ersten Primer, der mit dem zweiten Strang der doppelsträngigen Desoxyribonukleinsäure mit dem zweiten Allel hybridisiert ist;
(d) Aussetzen der Amplifikationsprodukte der Polymerasekettenreaktion zu Ionenaustauschchromatographie, wobei
der Größenunterschied des Amplifikationsprodukts der Polymerasekettenreaktion von dem ersten Primer und dem dritten Primer und dem Amplifikationsprodukt der Polymerasekettenreaktion von dem zweiten Primer und dem dritten Primer 0 Basenpaar, 1 Basenpaar, 2 Basenpaare, 3 Basenpaare, 4 Basenpaare, 5 Basenpaare, 6 Basenpaare, 7 Basenpaare, 8 Basenpaare, 9 Basenpaare oder 10 Basenpaare beträgt; und
(e) Nachweisen der Anwesenheit von einem oder beiden der ersten und zweiten Allele, basierend auf Elutionspositionen oder Elutionszeiten der Amplifikationsprodukte,
wobei der nicht-Nukleotid-Bestanteil eine Substanz ist, die eine Veränderung der elektrischen Ladung an dem 5'-Ende des Primers bewirkt.
(a) fournir un échantillon contenant un acide désoxyribonucléique double brin contenant un site polymorphe ;
(b) fournir une première amorce, une deuxième amorce et une troisième amorce, dans lequel
la séquence de la première amorce est complémentaire du second brin de l'acide désoxyribonucléique double brin présentant un premier allèle au niveau du site polymorphe, et une ou deux ou trois bases quelconques parmi trois bases au niveau de l'extrémité 3' ou une ou les deux parmi deux bases au niveau de l'extrémité 3' de la séquence de la première amorce correspond(ent) au site de polymorphisme, dans lequel
la séquence de la seconde amorce est complémentaire du second brin de l'acide désoxyribonucléique double brin présentant un second allèle au niveau du site polymorphe, et une ou deux ou trois bases quelconques parmi trois bases au niveau de l'extrémité 3' ou une ou les deux parmi deux bases au niveau de l'extrémité 3' de la séquence de la deuxième amorce correspond(ent) au site de polymorphisme, dans lequel
la séquence de la troisième amorce n'inclut pas le site polymorphe et est complémentaire du premier brin de l'acide désoxyribonucléique double brin, dans lequel
un composant non nucléotidique est ajouté à au moins l'une de la première amorce et de la deuxième amorce ;
(c) effectuer une réaction en chaîne par polymérase, dans lequel
la réaction en chaîne par polymérase est réalisée dans des conditions telles qu'un allongement de brin dû à une polymérase de la première amorce hybridée au second brin de l'acide désoxyribonucléique double brin présentant le premier allèle se produit préférentiellement par comparaison à un allongement de brin dû à une polymérase de la deuxième amorce hybridée au second brin de l'acide désoxyribonucléique double brin présentant le premier allèle, et en ce que l'allongement de brin dû à une polymérase de la deuxième amorce hybridée au second brin de l'acide désoxyribonucléique double brin présentant le second allèle se produit préférentiellement par comparaison à un allongement de brin dû à une polymérase de la première amorce hybridée au second brin de l'acide désoxyribonucléique double brin présentant le second allèle ;
(d) soumettre des produits d'amplification de la réaction en chaîne par polymérase à une chromatographie par échange d'ions, dans lequel
la différence de taille du produit d'amplification de la réaction en chaîne par polymérase à partir de la première amorce et de la troisième amorce et du produit d'amplification de la réaction en chaîne par polymérase à partir de la deuxième amorce et de la troisième amorce est de 0 paire de bases, 1 paire de bases, 2 paires de bases, 3 paires de bases, 4 paires de bases, 5 paires de bases, 6 paires de bases, 7 paires de bases, 8 paires de bases, 9 paires de bases ou 10 paires de bases ; et
(e) détecter la présence de l'un ou des deux des premier et second allèles sur la base de positions d'élution ou de temps d'élution des produits d'amplification,
dans lequel le composant non nucléotidique est une substance induisant un changement de charge électrique à l'extrémité 5' de l'amorce.
REFERENCES CITED IN THE DESCRIPTION
Patent documents cited in the description
Non-patent literature cited in the description