CA3004060C - Labeled deoxynucleoside triphosphates, uses thereof and methods for their preparation - Google Patents
Labeled deoxynucleoside triphosphates, uses thereof and methods for their preparation Download PDFInfo
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- CA3004060C CA3004060C CA3004060A CA3004060A CA3004060C CA 3004060 C CA3004060 C CA 3004060C CA 3004060 A CA3004060 A CA 3004060A CA 3004060 A CA3004060 A CA 3004060A CA 3004060 C CA3004060 C CA 3004060C
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- group
- compound
- disulfide
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- deoxynucleoside triphosphate
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- 239000001226 triphosphate Substances 0.000 title claims abstract description 145
- 235000011178 triphosphate Nutrition 0.000 title claims abstract description 143
- 238000000034 method Methods 0.000 title claims abstract description 68
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 title abstract description 10
- 238000002360 preparation method Methods 0.000 title description 4
- 150000001875 compounds Chemical class 0.000 claims abstract description 234
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 178
- 239000000203 mixture Substances 0.000 claims abstract description 92
- 125000006239 protecting group Chemical group 0.000 claims abstract description 83
- 238000012163 sequencing technique Methods 0.000 claims abstract description 52
- 125000005647 linker group Chemical group 0.000 claims description 158
- 238000006243 chemical reaction Methods 0.000 claims description 127
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 126
- -1 disulfide allyl Chemical group 0.000 claims description 114
- 239000000975 dye Substances 0.000 claims description 100
- 239000011541 reaction mixture Substances 0.000 claims description 88
- 125000000217 alkyl group Chemical group 0.000 claims description 75
- 125000002228 disulfide group Chemical group 0.000 claims description 60
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 54
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 50
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 49
- 150000007523 nucleic acids Chemical class 0.000 claims description 47
- 108020004707 nucleic acids Proteins 0.000 claims description 45
- 102000039446 nucleic acids Human genes 0.000 claims description 45
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 43
- 239000002777 nucleoside Substances 0.000 claims description 40
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 claims description 38
- 125000003118 aryl group Chemical group 0.000 claims description 33
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 28
- 229960002685 biotin Drugs 0.000 claims description 27
- 235000020958 biotin Nutrition 0.000 claims description 27
- 239000011616 biotin Substances 0.000 claims description 27
- 238000012546 transfer Methods 0.000 claims description 27
- 150000001412 amines Chemical class 0.000 claims description 26
- 150000001540 azides Chemical class 0.000 claims description 23
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 23
- VEVZDKMDCHTMIA-UHFFFAOYSA-N oxadithietane Chemical compound O1CSS1 VEVZDKMDCHTMIA-UHFFFAOYSA-N 0.000 claims description 20
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 claims description 19
- 125000001494 2-propynyl group Chemical group [H]C#CC([H])([H])* 0.000 claims description 12
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims description 11
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims description 11
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 claims description 10
- 229910052698 phosphorus Inorganic materials 0.000 claims description 9
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 claims description 8
- 230000006820 DNA synthesis Effects 0.000 claims description 7
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 claims description 5
- 125000003636 chemical group Chemical group 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000003797 solvolysis reaction Methods 0.000 claims description 3
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 3
- 229910006069 SO3H Inorganic materials 0.000 claims description 2
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 172
- XJLXLCWHYSPAJZ-UHFFFAOYSA-N dithiirane Chemical compound C1SS1 XJLXLCWHYSPAJZ-UHFFFAOYSA-N 0.000 abstract description 35
- 238000005516 engineering process Methods 0.000 abstract description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 205
- 125000003729 nucleotide group Chemical group 0.000 description 158
- 239000002773 nucleotide Substances 0.000 description 132
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 116
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 94
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 89
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 88
- 239000000243 solution Substances 0.000 description 66
- 239000013615 primer Substances 0.000 description 65
- 239000000047 product Substances 0.000 description 60
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 56
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 53
- 238000003776 cleavage reaction Methods 0.000 description 48
- 239000000741 silica gel Substances 0.000 description 46
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- 101150041968 CDC13 gene Proteins 0.000 description 43
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 43
- 230000007017 scission Effects 0.000 description 41
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 41
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 36
- 239000012299 nitrogen atmosphere Substances 0.000 description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- 238000005160 1H NMR spectroscopy Methods 0.000 description 30
- 238000003818 flash chromatography Methods 0.000 description 29
- 238000003756 stirring Methods 0.000 description 29
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 27
- 238000009396 hybridization Methods 0.000 description 26
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 24
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 24
- 238000002390 rotary evaporation Methods 0.000 description 24
- 239000007787 solid Substances 0.000 description 23
- 235000000346 sugar Nutrition 0.000 description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 22
- 239000003153 chemical reaction reagent Substances 0.000 description 22
- 239000007832 Na2SO4 Substances 0.000 description 21
- 229910052938 sodium sulfate Inorganic materials 0.000 description 21
- 235000011152 sodium sulphate Nutrition 0.000 description 21
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 20
- 239000000872 buffer Substances 0.000 description 19
- 238000004128 high performance liquid chromatography Methods 0.000 description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- WQOXQRCZOLPYPM-UHFFFAOYSA-N dimethyl disulfide Chemical compound CSSC WQOXQRCZOLPYPM-UHFFFAOYSA-N 0.000 description 18
- 239000003039 volatile agent Substances 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 16
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 16
- 239000003638 chemical reducing agent Substances 0.000 description 16
- 239000012153 distilled water Substances 0.000 description 16
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 16
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 15
- 238000005481 NMR spectroscopy Methods 0.000 description 15
- 229910052799 carbon Inorganic materials 0.000 description 15
- 238000010348 incorporation Methods 0.000 description 15
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 14
- HGCIXCUEYOPUTN-UHFFFAOYSA-N cyclohexene Chemical compound C1CCC=CC1 HGCIXCUEYOPUTN-UHFFFAOYSA-N 0.000 description 14
- 238000000746 purification Methods 0.000 description 14
- 238000004440 column chromatography Methods 0.000 description 13
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical class C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 13
- 150000002148 esters Chemical class 0.000 description 13
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- 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 description 12
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 12
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical class O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 12
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- 239000002808 molecular sieve Substances 0.000 description 12
- 229910052757 nitrogen Inorganic materials 0.000 description 12
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 12
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- 229920001971 elastomer Polymers 0.000 description 10
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethanethiol Chemical compound CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 10
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 10
- XYFCBTPGUUZFHI-UHFFFAOYSA-N phosphine group Chemical group P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 10
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- 125000004429 atom Chemical group 0.000 description 7
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- 238000001514 detection method Methods 0.000 description 7
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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| TWI639614B (zh) * | 2015-11-06 | 2018-11-01 | 美商齊亞雋科學公司 | 核苷酸類似物 |
| US11266673B2 (en) | 2016-05-23 | 2022-03-08 | The Trustees Of Columbia University In The City Of New York | Nucleotide derivatives and methods of use thereof |
| WO2018129644A1 (zh) * | 2017-01-10 | 2018-07-19 | 深圳华大智造科技有限公司 | 一种提高核酸聚合测序质量的方法、反应体系和试剂盒 |
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| CN119639879A (zh) | 2017-03-28 | 2025-03-18 | 哥伦比亚大学董事会 | 用于连接荧光标记与用于dna合成测序的碱基的具有不同可裂解连接子的3'-o-改性的核苷酸类似物 |
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| WO2019178393A1 (en) | 2018-03-15 | 2019-09-19 | The Trustees Of Columbia University In The City Of New York | Nucleotide analogues and use thereof for nucleic acid sequencing and analysis |
| WO2020056044A1 (en) | 2018-09-11 | 2020-03-19 | Singular Genomics Systems, Inc. | Modified archaeal family b polymerases |
| WO2020069424A1 (en) | 2018-09-28 | 2020-04-02 | Centrillion Technologies, Inc. | Disulfide-linked reversible terminators |
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| WO2021067970A1 (en) * | 2019-10-04 | 2021-04-08 | Centrillion Technologies, Inc. | Reversible terminators for dna sequencing and methods of using the same |
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| US11174281B1 (en) | 2020-04-24 | 2021-11-16 | Singular Genomics Systems, Inc. | Modified nucleotides and uses thereof |
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| CN115960152A (zh) * | 2022-10-11 | 2023-04-14 | 河南师范大学 | 一种炔丙醚基取代tpt3型非天然碱基三磷酸及其合成方法和应用 |
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