WO2007104834A1 - Substrats terminateurs pour adn polymérases - Google Patents
Substrats terminateurs pour adn polymérases Download PDFInfo
- Publication number
- WO2007104834A1 WO2007104834A1 PCT/FI2007/050127 FI2007050127W WO2007104834A1 WO 2007104834 A1 WO2007104834 A1 WO 2007104834A1 FI 2007050127 W FI2007050127 W FI 2007050127W WO 2007104834 A1 WO2007104834 A1 WO 2007104834A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lanthanide
- substrate according
- dna
- terminating
- lll
- Prior art date
Links
- 239000000758 substrate Substances 0.000 title claims abstract description 27
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 title claims abstract description 13
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 title claims abstract description 13
- 150000002602 lanthanoids Chemical class 0.000 claims abstract description 28
- 229910052747 lanthanoid Inorganic materials 0.000 claims abstract description 25
- 239000001226 triphosphate Substances 0.000 claims abstract description 14
- 235000011178 triphosphate Nutrition 0.000 claims abstract description 10
- 239000002777 nucleoside Substances 0.000 claims abstract description 6
- -1 nucleoside triphosphates Chemical class 0.000 claims abstract description 6
- 239000013522 chelant Substances 0.000 claims description 15
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 8
- 229910052692 Dysprosium Inorganic materials 0.000 claims description 7
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 claims description 6
- 229910052771 Terbium Inorganic materials 0.000 claims description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 6
- 125000002015 acyclic group Chemical group 0.000 claims description 6
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 6
- 229910052693 Europium Inorganic materials 0.000 claims description 5
- 229910052772 Samarium Inorganic materials 0.000 claims description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 4
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium atom Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 claims description 4
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 4
- OLXZPDWKRNYJJZ-UHFFFAOYSA-N 5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-ol Chemical compound C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(CO)O1 OLXZPDWKRNYJJZ-UHFFFAOYSA-N 0.000 claims description 3
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 claims description 3
- 238000001712 DNA sequencing Methods 0.000 claims description 3
- 229940104302 cytosine Drugs 0.000 claims description 3
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical group [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 3
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 claims description 3
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 claims description 3
- 229940035893 uracil Drugs 0.000 claims description 3
- LFRDGHVRPSURMV-YFKPBYRVSA-N (4s)-4,5-dihydroxypentanal Chemical group OC[C@@H](O)CCC=O LFRDGHVRPSURMV-YFKPBYRVSA-N 0.000 claims description 2
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 claims description 2
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 claims description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 claims description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- MENFBZNRKXAVOJ-UHFFFAOYSA-N azane;n,n-dibutylbutan-1-amine Chemical compound N.CCCCN(CCCC)CCCC MENFBZNRKXAVOJ-UHFFFAOYSA-N 0.000 claims description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 229910052744 lithium Inorganic materials 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- 229910052749 magnesium Inorganic materials 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 150000003512 tertiary amines Chemical class 0.000 claims description 2
- 150000003568 thioethers Chemical class 0.000 claims description 2
- 229940104230 thymidine Drugs 0.000 claims description 2
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 2
- 125000005647 linker group Chemical group 0.000 claims 3
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical class CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 11
- 239000002585 base Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 0 *C[C@]1O[C@@](*)CC1 Chemical compound *C[C@]1O[C@@](*)CC1 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000002285 radioactive effect Effects 0.000 description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Natural products O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 125000006853 reporter group Chemical group 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 230000006820 DNA synthesis Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 125000000837 carbohydrate group Chemical group 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 229910021644 lanthanide ion Inorganic materials 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- GGLAGENYOZKOMR-UHFFFAOYSA-N 2-[[6-[[bis(carboxymethyl)amino]methyl]pyridin-2-yl]methyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)CC1=CC=CC(CN(CC(O)=O)CC(O)=O)=N1 GGLAGENYOZKOMR-UHFFFAOYSA-N 0.000 description 1
- 125000006321 2-propynyl amino group Chemical group [H]C#CC([H])([H])N([H])* 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- ZECGAMULKVIREL-HOGWDWRMSA-N CC[C@H](C(C)C1(C)C)O[C@H]1N(C=CC(N)=N1)C1=O Chemical compound CC[C@H](C(C)C1(C)C)O[C@H]1N(C=CC(N)=N1)C1=O ZECGAMULKVIREL-HOGWDWRMSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N methyl monoether Natural products COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- SONNWYBIRXJNDC-VIFPVBQESA-N phenylephrine Chemical compound CNC[C@H](O)C1=CC=CC(O)=C1 SONNWYBIRXJNDC-VIFPVBQESA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 239000002718 pyrimidine nucleoside Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 150000003254 radicals Chemical group 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 229960003636 vidarabine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
Definitions
- This invention relates to novel derivatives of labeled nucleoside triphosphates suitable for DNA sequencing.
- DNA fragments are syn- thesised by DNA polymerase which incorporates deoxynucleotide monomers into a polymeric complementary copy of a template DNA strand.
- An oligonucleotide primer is used to initiate the synthesis of the new DNA stand from the template DNA at a single specific location (Sanger, F., Nicken, S., Coulson, A.R., 1977, PNAS, 74, 5463).
- ddNTP 2',3 ' -dideoxynucleoside 5 ' -triphosphate
- dNTP 2',3 ' -dideoxynucleoside 5 ' -triphosphate
- dNTP 2 ' -deoxynucleoside 5'- triphosphate
- the dideoxynucleotide is not able to form a phosphodiester bond with the next incoming dNTP, and the growth of that particular DNA chain stops.
- a series of strands is obtained, the lengths of which depend on the location of ddNTP.
- each fragment must be labelled in some manner.
- labelling has been accomplished with radioisotopes, such as 32 P or 35 S prior of during the polymerase reaction, i.e. using either a radioisotopically labelled primer of dNTPs.
- radioactive detection is very sensitive, it has intrinsic hazard, expense and problems associated with the short half-lives of the radioactive isotopes commonly used.
- a primer labelled with a detectable group such as a chemiluminescent dye
- a detectable group such as a chemiluminescent dye
- a chemiluminescent dye Heunkapiller, T., Kaiser, R.J., Koop, B. F., Hood, K.L, 1991 , Science, 254, 59, Smith, LM., Fung, S, Hunkapiller, M.W., Hunkapiller, T.J., Hood, L.E., 1985, Nucleic Acids Res., 13, 2399, Smith, L.M., Sanders, J.Z., Kaiser, R.J., Hughes, P., Dodd, C, Connell, C.R., Heiner, C, Kent, S.B.H., Hood, LE., 1986, Nature, 321 , 674);
- the labeled dNTPs ought to be almost as good substrates to DNA polymerases as normal dNTPs. Since DNA polymerases are very sensitive to structural changes of their substrates, the selection of methods to attach non-radioactive markers into dNTPs are rather limited. It has already shown that the well-known terminators of DNA synthesis, 2 ' ,3 ' -dideoxy-3 ' -amino NTPs (Chidzeavadze, Z.G., Beabealashvilli, R.
- the label is attached covalently to the nucleobase via a rigid propargylamine linker at C5 of the pyrimidine nucleosides and at C7 of 7-deazapurine nucleosides, e.g. at positions which are not involved in the formation of normal Watson-Crick base pairs.
- these label molecules used are organic dyes which suffer from commonly known drawbacks such as Raman scattering, concentration quenching and low water solubility.
- minisequencing A solid-phase method, called minisequencing, has been introduced for detection of point mutation of DNA (Syvanen, A.-C, Allto-Setala, K., Harju, L., S ⁇ deriund, H., 1990, Genomics, 8, 684, Jalanko, A., Kere, J., Savilahti, E., Schwartz, M., Syvanen, A.-C, S ⁇ deriund, H., 1992, Clin.
- lanthanide(lll) chelates such as strong long-decay time luminescence make them ideal markers for numerous applications. Furthermore, large Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Sm, Dy) in the analysis. Time resolved fluorimetric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening.
- the heterogenous DELFIA technique (EP 0139675 Bl; US 4,808,541; EP 0298939 Bl; US 6,127,529; US 4,565,790; WO 03/076939; Hemmila I., Dakubu, S., Mukkala, V.-M., Siitari, H., Lovgren, T., 1984, Anal. Biochem. 137, 335; Fl Pat. Appl. 20065030) is applied in assays requiring exceptional sensitivity, robustness and multi-label approach.
- the development highly stable and luminescent lanthanide(lll) chelates (Hemmila, I.; Mukkala, V.-M. 2001 , Crit. Rev. Clin. Lab. Sci.
- the main object of the present invention is to provide nucleoside 5 ' -triphosphates or their acylic analogues labeled with luminescent or non-luminescent lanthanide(lll) chelates to serve as terminating substrates for DNA polymerases.
- the major advantages of the present invention are:
- Each DNA nucleobase is detected by its own metal chelate, i.e. four bases (Ade, Gua, Cyt, Thy) and four metal chelates (Eu 3+ , Tb 3+ , Sm 3+ , Dy 3+ ). Accordingly, the base sequence can be analyzed by detecting the specific signal derived form the appropriate lanthanide chelate.
- the lanthanide(lll) chelate can be either luminescent or non-luminescent. Accordingly, the structure of the chelate can be chosen to fulfil the requirements of the desired detection technology.
- the sequencing procedure includes electrophoretic separation, it is desirable that the net charges of the terminating substrates do not differ from the natural 2'-deoxyribonucleoside 5 ' -triphosphates. In these cases, the use of neutral lanthanide chelates is advantageous.
- the present invention concerns a terminating substrate for DNA polymerases of formula (I)
- Z is triphosphate anion or its organic or inorganic salt
- R is a recognizing moiety, which is a nucleoside comprising a base bound to a 2,3-dideoxyribose or its acyclic derivative; and Z-R has the formula (II)
- ribose ring optionally is replaced by an acyclic derivative in which one or both of the 2- and 3- carbon atoms from the ribose ring optionally are missing;
- -L- is a linker
- X is a lantha ⁇ ide(lll) chelate.
- the invention concerns the use of the terminating substrate according to claim 1 in DNA sequencing for deter- mining the base sequence wherein each base is identified by detecting the signal derived from the ianthanide chelate of said terminating substrate.
- the base in the recognizing moiety R is adenine, guanine, cytosine, thymine or uracil. Also modifications of said bases can be used. As preferable modifications can be mentioned 7-deaza-adenine and 7- deazaguanine.
- a preferable acyclic derivative is the dimethyl ether bridge derived from the ribose ring.
- the salt is most preferably sodium, lithium, potassium, calcium, magnesium, ammonium tributylammonium or triethylammonium salt.
- the recognizing moiety R is a radical selected from the following seven structures;
- n 0 or 1.
- the linker -L- is connected to C7 of 7-deazaadenine, C7 of 7-deazaguanine, C5 of cytosine, C5 of uracil, C3 ' of 2 ' -deoxyguanosine, C3 ' 2 ' -deoxyadenosine, C3 ' of 2'- deoxycytidine or C3 ' of thymidine.
- the lanthanide chelate X is either luminescent or non-luminescent, and the lanthanide(lll) ion is selected in such a way that each DNA nucleobase referes to one lanthanide(lll) ion.
- any luminescent or non-luminescent lanthanide (III) chelate is suitable as reporter group in the terminating substrate according to the present invention
- the lanthanide chelates disclosed in the Background section above are preferable.
- Particularly preferable lanthanide (III) chelates for this purpose are luminescent chelates based on triazacycloalkanes and nonluminescent chelates based on pyridine-2,6-diyl- bis(methylenenitrilo)tetrakis(acetic acid).
- Biotinylated oligonucleotides and detection primer were purchased from Sigma-Genosys. Poymerase Pol B, dilution buffer, reaction buffer and acycloterminators were products of NEN. Streptavidin coated microtiter plates, assay buffer, wash buffer, enhancement solution, DELFIA Enhancer and lanthanide chelates were from PerkinElmer Life and Analytical Sciences. Minisequencing assays were analyzed with Victor multilabel oounter (Wallac Oy 1 PerkinElmer Life and Analytical Sciences).
- Biotinylated oligonucleotides (6 pmol/weil in 40 ⁇ L) were captured on streptavidin coated microtiter plates in 30 min by shaking at RT. To remove unbound templates wells were rinsed 4 times with wash buffer. r0041l Example 3. Minisequencing reactions and analysis
- Reaction mixture (40 ⁇ f) contained the detection primer (0.5- 5 pmol/well), polymerase Pol B (0.1 U/weil) and lanthanide chelate labeled acycloterminator (3a-d).
- the amount of the triphosphates depended on the lanthanide chelate and was as follows.
- the amount of Eu-GTP and Tb-UTP was 0.5 pmol/well and both Sm-ATP and Dy-CTP was 50 pmol/well.
- the reaction was allowed to proceed with shaking 20 minutes at 68°C. After the reactions wells were rinsed 6 times with wash buffer.
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Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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GB0818768A GB2450454B (en) | 2006-03-13 | 2007-03-08 | Terminating substrates for DNA polymerases |
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FI20065162A FI20065162A0 (fi) | 2006-03-13 | 2006-03-13 | Terminoivia DNA polymeraasisubstraatteja |
FI20065162 | 2006-03-13 |
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WO2007104834A1 true WO2007104834A1 (fr) | 2007-09-20 |
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PCT/FI2007/050127 WO2007104834A1 (fr) | 2006-03-13 | 2007-03-08 | Substrats terminateurs pour adn polymérases |
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FI (1) | FI20065162A0 (fr) |
GB (1) | GB2450454B (fr) |
WO (1) | WO2007104834A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8119800B2 (en) | 2007-12-21 | 2012-02-21 | Korea Research Institute Of Chemical Technology | Processes for preparing HIV reverse transcriptase inhibitors |
US8334295B2 (en) | 2007-06-29 | 2012-12-18 | Korea Research Institute Of Chemical Technology | Pyrimidine derivatives as HIV reverse transcriptase inhibitors |
US8354421B2 (en) | 2007-06-29 | 2013-01-15 | Korea Research Insitute Of Chemical Technology | HIV reverse transcriptase inhibitors |
US9745253B2 (en) | 2015-03-13 | 2017-08-29 | Forma Therapeutics, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
Citations (7)
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EP0251786A2 (fr) * | 1986-07-02 | 1988-01-07 | E.I. Du Pont De Nemours And Company | Alkynylaminonucléotides |
EP0340675A2 (fr) * | 1988-05-02 | 1989-11-08 | The Perkin-Elmer Corporation | Procédé de dosage de nucléotides marquées avec lanthanide par fluorométrie à résolution temporelle |
WO1990000623A1 (fr) * | 1988-07-08 | 1990-01-25 | Wallac Oy | Analyse par fluorescence a resolution temporelle et marquage multiple de sequences d'acide nucleique, a l'aide de chelates de lanthanide |
US5798210A (en) * | 1993-03-26 | 1998-08-25 | Institut Pasteur | Derivatives utilizable in nucleic acid sequencing |
US6255475B1 (en) * | 1995-01-31 | 2001-07-03 | Marek Kwiatkowski | Chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation |
US20050084451A1 (en) * | 2003-08-29 | 2005-04-21 | Wallac Oy | Novel chelating agents and chelates and their use |
US20050181393A1 (en) * | 2003-12-18 | 2005-08-18 | Wallac Oy | Novel chelating agents and highly luminescent and stable chelates and their use |
-
2006
- 2006-03-13 FI FI20065162A patent/FI20065162A0/fi not_active Application Discontinuation
-
2007
- 2007-03-08 GB GB0818768A patent/GB2450454B/en not_active Expired - Fee Related
- 2007-03-08 WO PCT/FI2007/050127 patent/WO2007104834A1/fr active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0251786A2 (fr) * | 1986-07-02 | 1988-01-07 | E.I. Du Pont De Nemours And Company | Alkynylaminonucléotides |
EP0340675A2 (fr) * | 1988-05-02 | 1989-11-08 | The Perkin-Elmer Corporation | Procédé de dosage de nucléotides marquées avec lanthanide par fluorométrie à résolution temporelle |
WO1990000623A1 (fr) * | 1988-07-08 | 1990-01-25 | Wallac Oy | Analyse par fluorescence a resolution temporelle et marquage multiple de sequences d'acide nucleique, a l'aide de chelates de lanthanide |
US5798210A (en) * | 1993-03-26 | 1998-08-25 | Institut Pasteur | Derivatives utilizable in nucleic acid sequencing |
US6255475B1 (en) * | 1995-01-31 | 2001-07-03 | Marek Kwiatkowski | Chain terminators, the use thereof for nucleic acid sequencing and synthesis and a method of their preparation |
US20050084451A1 (en) * | 2003-08-29 | 2005-04-21 | Wallac Oy | Novel chelating agents and chelates and their use |
US20050181393A1 (en) * | 2003-12-18 | 2005-08-18 | Wallac Oy | Novel chelating agents and highly luminescent and stable chelates and their use |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8334295B2 (en) | 2007-06-29 | 2012-12-18 | Korea Research Institute Of Chemical Technology | Pyrimidine derivatives as HIV reverse transcriptase inhibitors |
US8354421B2 (en) | 2007-06-29 | 2013-01-15 | Korea Research Insitute Of Chemical Technology | HIV reverse transcriptase inhibitors |
US8119800B2 (en) | 2007-12-21 | 2012-02-21 | Korea Research Institute Of Chemical Technology | Processes for preparing HIV reverse transcriptase inhibitors |
US9745253B2 (en) | 2015-03-13 | 2017-08-29 | Forma Therapeutics, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
US10266487B2 (en) | 2015-03-13 | 2019-04-23 | Forma Therapeutics, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
US10508077B2 (en) | 2015-03-13 | 2019-12-17 | Forma Therapeutics, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
US10988441B2 (en) | 2015-03-13 | 2021-04-27 | Valo Early Discovery, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
US11919839B2 (en) | 2015-03-13 | 2024-03-05 | Valo Health, Inc. | Alpha-cinnamide compounds and compositions as HDAC8 inhibitors |
Also Published As
Publication number | Publication date |
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GB2450454A (en) | 2008-12-24 |
GB2450454B (en) | 2011-04-13 |
GB0818768D0 (en) | 2008-11-19 |
FI20065162A0 (fi) | 2006-03-13 |
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