WO2018129214A1 - Stepwise sequencing by non-labeled reversible terminators or natural nucleotides - Google Patents
Stepwise sequencing by non-labeled reversible terminators or natural nucleotides Download PDFInfo
- Publication number
- WO2018129214A1 WO2018129214A1 PCT/US2018/012425 US2018012425W WO2018129214A1 WO 2018129214 A1 WO2018129214 A1 WO 2018129214A1 US 2018012425 W US2018012425 W US 2018012425W WO 2018129214 A1 WO2018129214 A1 WO 2018129214A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- affinity reagent
- affinity
- nucleobase
- incorporated
- reversible terminator
- Prior art date
Links
- 230000002441 reversible effect Effects 0.000 title claims abstract description 212
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 128
- 125000003729 nucleotide group Chemical group 0.000 title claims description 252
- 239000002773 nucleotide Substances 0.000 title claims description 195
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 500
- 238000000034 method Methods 0.000 claims abstract description 198
- 230000027455 binding Effects 0.000 claims abstract description 133
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 88
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 82
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 82
- 238000010348 incorporation Methods 0.000 claims abstract description 48
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 32
- 230000000903 blocking effect Effects 0.000 claims description 283
- 108020004414 DNA Proteins 0.000 claims description 122
- 239000013615 primer Substances 0.000 claims description 114
- 239000005547 deoxyribonucleotide Substances 0.000 claims description 91
- -1 azidomethyl Chemical group 0.000 claims description 88
- 125000002637 deoxyribonucleotide group Chemical group 0.000 claims description 88
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 70
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 58
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 56
- 238000006243 chemical reaction Methods 0.000 claims description 48
- 235000000346 sugar Nutrition 0.000 claims description 42
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 30
- 102000053602 DNA Human genes 0.000 claims description 29
- 230000000295 complement effect Effects 0.000 claims description 29
- 229930024421 Adenine Natural products 0.000 claims description 28
- 229960000643 adenine Drugs 0.000 claims description 28
- 229940104302 cytosine Drugs 0.000 claims description 28
- 229940113082 thymine Drugs 0.000 claims description 28
- 239000003795 chemical substances by application Substances 0.000 claims description 26
- 239000007850 fluorescent dye Substances 0.000 claims description 23
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 20
- 125000004404 heteroalkyl group Chemical group 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 15
- 229910019142 PO4 Inorganic materials 0.000 claims description 14
- 235000021317 phosphate Nutrition 0.000 claims description 14
- 239000001226 triphosphate Substances 0.000 claims description 13
- 235000011178 triphosphate Nutrition 0.000 claims description 13
- 125000003342 alkenyl group Chemical group 0.000 claims description 11
- 125000000217 alkyl group Chemical group 0.000 claims description 11
- 125000000304 alkynyl group Chemical group 0.000 claims description 10
- 229940035893 uracil Drugs 0.000 claims description 10
- 108020004635 Complementary DNA Proteins 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 claims description 7
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 125000003545 alkoxy group Chemical group 0.000 claims description 6
- 125000005282 allenyl group Chemical group 0.000 claims description 6
- 238000010804 cDNA synthesis Methods 0.000 claims description 6
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 claims description 6
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 6
- 125000004184 methoxymethyl group Chemical group [H]C([H])([H])OC([H])([H])* 0.000 claims description 5
- 125000006502 nitrobenzyl group Chemical group 0.000 claims description 5
- 239000003155 DNA primer Substances 0.000 claims description 4
- 125000002252 acyl group Chemical group 0.000 claims description 4
- YLFIGGHWWPSIEG-UHFFFAOYSA-N aminoxyl Chemical group [O]N YLFIGGHWWPSIEG-UHFFFAOYSA-N 0.000 claims description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 3
- 239000002987 primer (paints) Substances 0.000 claims description 3
- 125000005017 substituted alkenyl group Chemical group 0.000 claims description 3
- 125000004426 substituted alkynyl group Chemical group 0.000 claims description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims 3
- 238000000137 annealing Methods 0.000 claims 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 49
- 230000015572 biosynthetic process Effects 0.000 abstract description 19
- 238000013459 approach Methods 0.000 description 74
- 238000001514 detection method Methods 0.000 description 44
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 39
- 239000000047 product Substances 0.000 description 38
- 239000000427 antigen Substances 0.000 description 36
- 150000001875 compounds Chemical class 0.000 description 34
- 108091007433 antigens Proteins 0.000 description 33
- 102000036639 antigens Human genes 0.000 description 33
- 108090000623 proteins and genes Proteins 0.000 description 32
- 102000004169 proteins and genes Human genes 0.000 description 32
- 238000003491 array Methods 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 29
- 239000002777 nucleoside Substances 0.000 description 28
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 26
- 239000000975 dye Substances 0.000 description 26
- 230000003053 immunization Effects 0.000 description 26
- 241000283973 Oryctolagus cuniculus Species 0.000 description 25
- 238000002649 immunization Methods 0.000 description 25
- 125000005647 linker group Chemical group 0.000 description 24
- 102000040430 polynucleotide Human genes 0.000 description 24
- 108091033319 polynucleotide Proteins 0.000 description 24
- 239000002157 polynucleotide Substances 0.000 description 24
- 239000011541 reaction mixture Substances 0.000 description 22
- 230000009870 specific binding Effects 0.000 description 22
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 21
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 21
- 238000012986 modification Methods 0.000 description 21
- 230000004048 modification Effects 0.000 description 21
- 239000011347 resin Substances 0.000 description 20
- 229920005989 resin Polymers 0.000 description 20
- 108091034117 Oligonucleotide Proteins 0.000 description 19
- 239000011324 bead Substances 0.000 description 19
- 230000021615 conjugation Effects 0.000 description 19
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 19
- 108091023037 Aptamer Proteins 0.000 description 18
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 18
- 229940098773 bovine serum albumin Drugs 0.000 description 18
- 238000002372 labelling Methods 0.000 description 18
- 239000000126 substance Substances 0.000 description 18
- 239000000758 substrate Substances 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 125000004429 atom Chemical group 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 16
- 229940127073 nucleoside analogue Drugs 0.000 description 16
- 150000003833 nucleoside derivatives Chemical class 0.000 description 16
- 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 15
- 125000002467 phosphate group Chemical class [H]OP(=O)(O[H])O[*] 0.000 description 15
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 14
- 239000002953 phosphate buffered saline Substances 0.000 description 14
- 108010025905 Cystine-Knot Miniproteins Proteins 0.000 description 13
- 239000000872 buffer Substances 0.000 description 13
- 238000011534 incubation Methods 0.000 description 13
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 13
- 230000008569 process Effects 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 230000003321 amplification Effects 0.000 description 12
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 12
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 12
- 230000002163 immunogen Effects 0.000 description 12
- 238000003199 nucleic acid amplification method Methods 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 229920000936 Agarose Polymers 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 10
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 10
- 238000007792 addition Methods 0.000 description 10
- 150000001412 amines Chemical class 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 description 9
- 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 9
- 235000011180 diphosphates Nutrition 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 150000002148 esters Chemical class 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 241000283707 Capra Species 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- 239000002671 adjuvant Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 238000011033 desalting Methods 0.000 description 8
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 8
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 8
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 8
- 125000003835 nucleoside group Chemical group 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 108091028732 Concatemer Proteins 0.000 description 7
- 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 7
- 238000004458 analytical method Methods 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 239000011807 nanoball Substances 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 108091008108 affimer Proteins 0.000 description 6
- 238000001261 affinity purification Methods 0.000 description 6
- 239000011230 binding agent Substances 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 235000018977 lysine Nutrition 0.000 description 6
- 238000002493 microarray Methods 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 6
- 238000012175 pyrosequencing Methods 0.000 description 6
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 5
- 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 5
- 238000001712 DNA sequencing Methods 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 5
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000237988 Patellidae Species 0.000 description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound 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 5
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 5
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound 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 5
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 235000017557 sodium bicarbonate Nutrition 0.000 description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 5
- 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 description 4
- PFKFTWBEEFSNDU-UHFFFAOYSA-N 1,1'-Carbonyldiimidazole Substances C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 4
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 4
- 229930010555 Inosine Natural products 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 229960005305 adenosine Drugs 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 4
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 4
- CXVQSUBJMYZELD-UHFFFAOYSA-N ethyl 4-aminobutanoate;hydrochloride Chemical compound [Cl-].CCOC(=O)CCC[NH3+] CXVQSUBJMYZELD-UHFFFAOYSA-N 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 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 description 4
- 229910052731 fluorine Inorganic materials 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 229960003786 inosine Drugs 0.000 description 4
- 238000002823 phage display Methods 0.000 description 4
- 150000004713 phosphodiesters Chemical class 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000005096 rolling process Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 241000894007 species Species 0.000 description 4
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 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
- KOFLVDBWRHFSAB-UHFFFAOYSA-N 1,2,4,5-tetrahydro-1-(phenylmethyl)-5,9b(1',2')-benzeno-9bh-benz(g)indol-3(3ah)-one Chemical compound C1C(C=2C3=CC=CC=2)C2=CC=CC=C2C23C1C(=O)CN2CC1=CC=CC=C1 KOFLVDBWRHFSAB-UHFFFAOYSA-N 0.000 description 3
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical class C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 3
- 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 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 102000004594 DNA Polymerase I Human genes 0.000 description 3
- 108010017826 DNA Polymerase I Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108010047357 Luminescent Proteins Proteins 0.000 description 3
- 102000006830 Luminescent Proteins Human genes 0.000 description 3
- 108010079855 Peptide Aptamers Proteins 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 3
- 150000003838 adenosines Chemical class 0.000 description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 150000001718 carbodiimides Chemical class 0.000 description 3
- 150000001721 carbon Chemical class 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 125000000753 cycloalkyl group Chemical group 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 102000013415 peroxidase activity proteins Human genes 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 150000003141 primary amines Chemical class 0.000 description 3
- 150000003212 purines Chemical class 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000001022 rhodamine dye Substances 0.000 description 3
- 231100000241 scar Toxicity 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 229960002555 zidovudine Drugs 0.000 description 3
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 3
- NCMVOABPESMRCP-SHYZEUOFSA-N 2'-deoxycytosine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 NCMVOABPESMRCP-SHYZEUOFSA-N 0.000 description 2
- LTFMZDNNPPEQNG-KVQBGUIXSA-N 2'-deoxyguanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 LTFMZDNNPPEQNG-KVQBGUIXSA-N 0.000 description 2
- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical compound O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 2
- PGSPUKDWUHBDKJ-UHFFFAOYSA-N 6,7-dihydro-3h-purin-2-amine Chemical class C1NC(N)=NC2=C1NC=N2 PGSPUKDWUHBDKJ-UHFFFAOYSA-N 0.000 description 2
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- WSNMPAVSZJSIMT-UHFFFAOYSA-N COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 Chemical compound COc1c(C)c2COC(=O)c2c(O)c1CC(O)C1(C)CCC(=O)O1 WSNMPAVSZJSIMT-UHFFFAOYSA-N 0.000 description 2
- 101100217502 Caenorhabditis elegans lgg-3 gene Proteins 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- XCHARIIIZLLEBL-UHFFFAOYSA-N Medicagenic acid 3-O-beta-D-glucoside Chemical compound C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C(O)=O)C)(C)C1=CCC2C3(C)CC(O)C4OC1OC(CO)C(O)C(O)C1O XCHARIIIZLLEBL-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 2
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 2
- 241000276498 Pollachius virens Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 2
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 2
- 241001495444 Thermococcus sp. Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- WREGKURFCTUGRC-POYBYMJQSA-N Zalcitabine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 125000004104 aryloxy group Chemical group 0.000 description 2
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- JMXMXKRNIYCNRV-UHFFFAOYSA-N bis(hydroxymethyl)phosphanylmethanol Chemical compound OCP(CO)CO JMXMXKRNIYCNRV-UHFFFAOYSA-N 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000004671 cell-free system Anatomy 0.000 description 2
- 230000003196 chaotropic effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000000412 dendrimer Substances 0.000 description 2
- 229920000736 dendritic polymer Polymers 0.000 description 2
- KHWCHTKSEGGWEX-UHFFFAOYSA-N deoxyadenylic acid Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(O)=O)O1 KHWCHTKSEGGWEX-UHFFFAOYSA-N 0.000 description 2
- LTFMZDNNPPEQNG-UHFFFAOYSA-N deoxyguanylic acid Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(COP(O)(O)=O)O1 LTFMZDNNPPEQNG-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- VGONTNSXDCQUGY-UHFFFAOYSA-N desoxyinosine Natural products C1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 VGONTNSXDCQUGY-UHFFFAOYSA-N 0.000 description 2
- 150000002019 disulfides Chemical group 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 125000001072 heteroaryl group Chemical group 0.000 description 2
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 150000002972 pentoses Chemical group 0.000 description 2
- RDOWQLZANAYVLL-UHFFFAOYSA-N phenanthridine Chemical compound C1=CC=C2C3=CC=CC=C3C=NC2=C1 RDOWQLZANAYVLL-UHFFFAOYSA-N 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- AMFDITJFBUXZQN-KUBHLMPHSA-N (2s,3s,4r,5r)-2-(4-amino-5h-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol Chemical compound C=1NC=2C(N)=NC=NC=2C=1[C@@H]1N[C@H](CO)[C@@H](O)[C@H]1O AMFDITJFBUXZQN-KUBHLMPHSA-N 0.000 description 1
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 1
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- 125000006656 (C2-C4) alkenyl group Chemical group 0.000 description 1
- 125000006650 (C2-C4) alkynyl group Chemical group 0.000 description 1
- SLLFVLKNXABYGI-UHFFFAOYSA-N 1,2,3-benzoxadiazole Chemical class C1=CC=C2ON=NC2=C1 SLLFVLKNXABYGI-UHFFFAOYSA-N 0.000 description 1
- WKKCYLSCLQVWFD-UHFFFAOYSA-N 1,2-dihydropyrimidin-4-amine Chemical compound N=C1NCNC=C1 WKKCYLSCLQVWFD-UHFFFAOYSA-N 0.000 description 1
- BGGCPIFVRJFAKF-UHFFFAOYSA-N 1-[4-(1,3-benzoxazol-2-yl)phenyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1C1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 BGGCPIFVRJFAKF-UHFFFAOYSA-N 0.000 description 1
- RJKGJBPXVHTNJL-UHFFFAOYSA-N 1-nitronaphthalene Chemical group C1=CC=C2C([N+](=O)[O-])=CC=CC2=C1 RJKGJBPXVHTNJL-UHFFFAOYSA-N 0.000 description 1
- TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 1
- YKBGVTZYEHREMT-UHFFFAOYSA-N 2'-deoxyguanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(CO)O1 YKBGVTZYEHREMT-UHFFFAOYSA-N 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical class C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical class O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- HTFNVAVTYILUCF-UHFFFAOYSA-N 2-[2-ethoxy-4-[4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl]anilino]-5-methyl-11-methylsulfonylpyrimido[4,5-b][1,4]benzodiazepin-6-one Chemical compound CCOc1cc(ccc1Nc1ncc2N(C)C(=O)c3ccccc3N(c2n1)S(C)(=O)=O)C(=O)N1CCC(CC1)N1CCN(C)CC1 HTFNVAVTYILUCF-UHFFFAOYSA-N 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- BCHZICNRHXRCHY-UHFFFAOYSA-N 2h-oxazine Chemical compound N1OC=CC=C1 BCHZICNRHXRCHY-UHFFFAOYSA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
- YICAEXQYKBMDNH-UHFFFAOYSA-N 3-[bis(3-hydroxypropyl)phosphanyl]propan-1-ol Chemical compound OCCCP(CCCO)CCCO YICAEXQYKBMDNH-UHFFFAOYSA-N 0.000 description 1
- 125000000474 3-butynyl group Chemical group [H]C#CC([H])([H])C([H])([H])* 0.000 description 1
- KBDWGFZSICOZSJ-UHFFFAOYSA-N 5-methyl-2,3-dihydro-1H-pyrimidin-4-one Chemical compound N1CNC=C(C1=O)C KBDWGFZSICOZSJ-UHFFFAOYSA-N 0.000 description 1
- VTRBOZNMGVDGHY-UHFFFAOYSA-N 6-(4-methylanilino)naphthalene-2-sulfonic acid Chemical compound C1=CC(C)=CC=C1NC1=CC=C(C=C(C=C2)S(O)(=O)=O)C2=C1 VTRBOZNMGVDGHY-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- UKLNSYRWDXRTER-UHFFFAOYSA-N 7-isocyanato-3-phenylchromen-2-one Chemical compound O=C1OC2=CC(N=C=O)=CC=C2C=C1C1=CC=CC=C1 UKLNSYRWDXRTER-UHFFFAOYSA-N 0.000 description 1
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 1
- NLSUMBWPPJUVST-UHFFFAOYSA-N 9-isothiocyanatoacridine Chemical compound C1=CC=C2C(N=C=S)=C(C=CC=C3)C3=NC2=C1 NLSUMBWPPJUVST-UHFFFAOYSA-N 0.000 description 1
- 108010000239 Aequorin Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000025165 Autoerythrocyte sensitization syndrome Diseases 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- OJRUSAPKCPIVBY-KQYNXXCUSA-N C1=NC2=C(N=C(N=C2N1[C@H]3[C@@H]([C@@H]([C@H](O3)COP(=O)(CP(=O)(O)O)O)O)O)I)N Chemical compound C1=NC2=C(N=C(N=C2N1[C@H]3[C@@H]([C@@H]([C@H](O3)COP(=O)(CP(=O)(O)O)O)O)O)I)N OJRUSAPKCPIVBY-KQYNXXCUSA-N 0.000 description 1
- 125000006374 C2-C10 alkenyl group Chemical group 0.000 description 1
- 125000005865 C2-C10alkynyl group Chemical group 0.000 description 1
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 1
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 1
- 125000004648 C2-C8 alkenyl group Chemical group 0.000 description 1
- 125000004649 C2-C8 alkynyl group Chemical group 0.000 description 1
- 229910014585 C2-Ce Inorganic materials 0.000 description 1
- 101100454808 Caenorhabditis elegans lgg-2 gene Proteins 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-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
- 108020001019 DNA Primers Proteins 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102100029764 DNA-directed DNA/RNA polymerase mu Human genes 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- XQSPYNMVSIKCOC-NTSWFWBYSA-N Emtricitabine Chemical compound C1=C(F)C(N)=NC(=O)N1[C@H]1O[C@@H](CO)SC1 XQSPYNMVSIKCOC-NTSWFWBYSA-N 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 238000001327 Förster resonance energy transfer Methods 0.000 description 1
- 102000034286 G proteins Human genes 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 108010078851 HIV Reverse Transcriptase Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- XNKLLVCARDGLGL-JGVFFNPUSA-N Stavudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1C=C[C@@H](CO)O1 XNKLLVCARDGLGL-JGVFFNPUSA-N 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 0.000 description 1
- 108091008107 XNA aptamers Proteins 0.000 description 1
- 229960004748 abacavir Drugs 0.000 description 1
- MCGSCOLBFJQGHM-SCZZXKLOSA-N abacavir Chemical compound C=12N=CN([C@H]3C=C[C@@H](CO)C3)C2=NC(N)=NC=1NC1CC1 MCGSCOLBFJQGHM-SCZZXKLOSA-N 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 239000000999 acridine dye Substances 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 150000001251 acridines Chemical class 0.000 description 1
- IRLPACMLTUPBCL-FCIPNVEPSA-N adenosine-5'-phosphosulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@@H](CO[P@](O)(=O)OS(O)(=O)=O)[C@H](O)[C@H]1O IRLPACMLTUPBCL-FCIPNVEPSA-N 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 238000002819 bacterial display Methods 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229940125773 compound 10 Drugs 0.000 description 1
- 229940125758 compound 15 Drugs 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000392 cycloalkenyl group Chemical group 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960002656 didanosine Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 229960000366 emtricitabine Drugs 0.000 description 1
- 229960000980 entecavir Drugs 0.000 description 1
- YXPVEXCTPGULBZ-WQYNNSOESA-N entecavir hydrate Chemical compound O.C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)C1=C YXPVEXCTPGULBZ-WQYNNSOESA-N 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- LIYGYAHYXQDGEP-UHFFFAOYSA-N firefly oxyluciferin Natural products Oc1csc(n1)-c1nc2ccc(O)cc2s1 LIYGYAHYXQDGEP-UHFFFAOYSA-N 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 239000001021 fluorone dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- AFQIYTIJXGTIEY-UHFFFAOYSA-N hydrogen carbonate;triethylazanium Chemical compound OC(O)=O.CCN(CC)CC AFQIYTIJXGTIEY-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 229960004716 idoxuridine Drugs 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012804 iterative process Methods 0.000 description 1
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 1
- 229960001627 lamivudine Drugs 0.000 description 1
- JTEGQNOMFQHVDC-NKWVEPMBSA-N lamivudine Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1 JTEGQNOMFQHVDC-NKWVEPMBSA-N 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 235000019689 luncheon sausage Nutrition 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000002824 mRNA display Methods 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000012775 microarray technology Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000302 molecular modelling Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 150000005002 naphthylamines Chemical class 0.000 description 1
- 125000005184 naphthylamino group Chemical group C1(=CC=CC2=CC=CC=C12)N* 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 238000003499 nucleic acid array Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 229960001699 ofloxacin Drugs 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical compound S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 230000008832 photodamage Effects 0.000 description 1
- 230000010399 physical interaction Effects 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 150000003220 pyrenes Chemical class 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000985 reactive dye Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000002702 ribosome display Methods 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007841 sequencing by ligation Methods 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 235000019187 sodium-L-ascorbate Nutrition 0.000 description 1
- 239000011755 sodium-L-ascorbate Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 150000001629 stilbenes Chemical class 0.000 description 1
- 235000021286 stilbenes Nutrition 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229960005311 telbivudine Drugs 0.000 description 1
- IQFYYKKMVGJFEH-CSMHCCOUSA-N telbivudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1O[C@@H](CO)[C@H](O)C1 IQFYYKKMVGJFEH-CSMHCCOUSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- VSQQQLOSPVPRAZ-RRKCRQDMSA-N trifluridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(C(F)(F)F)=C1 VSQQQLOSPVPRAZ-RRKCRQDMSA-N 0.000 description 1
- 229960003962 trifluridine Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 229960003636 vidarabine Drugs 0.000 description 1
- 239000001018 xanthene dye Substances 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
- 229960000523 zalcitabine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
Definitions
- SBS sequencing- by-synthesis
- SBS requires the controlled (i.e., one at a time) incorporation of the correct complementary nucleotide opposite the oligonucleotide being sequenced. This allows for accurate sequencing by adding nucleotides in multiple cycles as each nucleotide residue is sequenced one at a time, thus preventing an uncontrolled series of incorporations occurring.
- RTs reversible terminator nucleotides
- each RT comprises a modified nucleotide that includes (1) a blocking group that ensures that only a single base can be added by a DNA polymerase enzyme to the 3' end of a growing DNA copy strand, and (2) a fluorescent label that can be detected by a camera.
- templates and sequencing primers are fixed to a solid support and the support is exposed to each of four DNA nucleotide analogs, each comprising a different fluorophore attached to the nitrogenous base by a cleavable linker, and a 3'-0-azidomethyl group at the 3'-OH position of deoxyribose, and DNA polymerase.
- RTs reversibly terminated dNTPs
- a label e.g., dye
- a cleavable linker resulting in a) a chemical scar left on the incorporated bases after label cleavage, b) less efficient incorporation, c) quenching, d) excited dye induced termination of extension, and reducing signal in each sequencing cycle.
- the present invention relates to methods and compositions for nucleic acid analysis and sequencing.
- an SBS sequencing method in which the last incorporated nucleotide base is identified by binding of an affinity reagent (e.g., antibody, aptamer, affimer, knottin, etc.) that recognizes the base, the sugar, a cleavable blocking group or a combination of these components in the last incorporated nucleotide.
- an affinity reagent e.g., antibody, aptamer, affimer, knottin, etc.
- the binding is directly or indirectly associated with production of a detectable signal.
- the invention provides methods of sequencing that employ non-labeled reversible terminator (NLRT) nucleotides.
- a reversible terminator (RT) nucleotide is a modified deoxynucleotide triphosphate (dNTP) or dNTP analog that contains a removable blocking group that ensures that only a single base can be added by a DNA polymerase enzyme to the 3' end of a growing DNA copy strand.
- dNTP 2'-deoxynucleoside triphosphates
- the incorporation of a dNTP (2'-deoxynucleoside triphosphates) to the 3' end of the growing strand during DNA synthesis involves the release of pyrophosphate, and when a dNTP is incorporated into a DNA strand the incorporated portion is a nucleotide monophosphate (or more precisely, a nucleotide monomer linked by phosphodiester bond(s) to one or two adjacent nucleotide monomers).
- a reversible terminator (RT) nucleotide is a modified deoxynucleotide triphosphate (dNTP) or dNTP analog that contains a removable blocking group that ensures that only a single base can be added by a DNA polymerase enzyme to the 3' end of a growing DNA copy strand.
- dNTP deoxynucleotide triphosphate
- a non-labeled RT nucleotide does not contain a detectable label.
- the nucleotide or nucleotide analogue is incorporated by a polymerase, extending the 3' end of the DNA copy strand by one base, and unincorporated nucleotides or nucleotide analogues are washed away.
- An affinity reagent is introduced that specifically recognizes and binds to an epitope(s) of the newly incorporated nucleotides or nucleotide analog. After an image is taken, the blocking group and the labeled affinity reagent are removed from the DNA, allowing the next cycle of sequencing to begin.
- the epitope recognized by the affinity reagent is formed by the incorporated nucleoside itself (that is, the base plus sugar) or the nucleoside and 3' blocking group.
- the epitope recognized by the affinity reagent is formed by the reversible terminator itself, the reversible terminator in combination with the deoxyribose, or the reversible terminator in combination with the nucleobase or nucleobase and deoxyribose.
- the present invention provides methods for sequencing a nucleic acid, comprising: (a) contacting a nucleic acid template comprising the nucleic acid, a nucleic acid primer complementary to a portion of said template, a polymerase, and an unlabeled RT of Formula I:
- R 1 is a 3'-0 reversible blocking group
- R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof
- R 3 comprises or consists of one or more phosphates
- the primer is extended to incorporate the unlabeled RT into a sequence complementary to the nucleic acid template, thereby producing an unlabeled extension product comprising the incorporated RT
- the nucleobase is conjugated to a cleavable linker that connects the base to a detectable label such as a fluorophore.
- a detectable label such as a fluorophore.
- R 2 is not a nucleobase conjugated to a dye or other detectable label by a linker.
- such a method further comprises (d) removing the reversible blocking group from the RT to produce a 3'-OH; and (e) removing the affinity reagent from the RT.
- such a method further comprises repeating steps of the method one or more times, that is, performing multiple cycles of sequencing, wherein at least a portion of the sequence of said nucleic acid template is determined.
- such a method comprises removing the reversible blocking group and the affinity reagent in the same reaction.
- such a method comprises removing the affinity reagent(s) without removing the reversible blocking group(s) and re-probing with difference affinity reagents.
- the affinity reagent may include antibodies (including binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, or any other known agent that binds an incorporated NLRT with a suitable specificity and affinity.
- the affinity reagent is an antibody.
- the affinity reagent is an antibody comprising detectable label that is a fluorescent label.
- R 1 is selected from the group consisting of ally I, azidomethyl, aminoalkoxyl, 2-cyanoethyl, substituted a Iky I, unsubstituted a Iky I, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heteroalkenyl, unsubstituted heteroalkenyl, substituted heteroalkynyl, unsubstituted heteroalkynyl, allenyl, cis-cyanoethenyl, trans- cyanoethenyl, cis-cyanofluoroethenyl, trans-cyanofluoroethenyl, cis-trifluoromethylethenyl, trans-trifluoromethylethenyl, biscyanoethenyl, bisfluor
- R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), and thymine (T).
- R 3 consists of or comprises one or more phosphates.
- non-labeled reversible terminator may refer to the triphosphate form of the nucleotide analog, or may refer to the incorporated NLRT.
- methods for sequencing a nucleic acid comprising: (a) providing a DNA array comprising (i) a plurality of template DNA molecules, each template DNA molecule comprising a fragment of the nucleic acid, wherein each of said plurality of template DNA molecules is attached at a position of the array, (b) contacting the DNA array with a nucleic acid primer complementary to a portion of each of said template DNA molecules, a polymerase, and an unlabeled RT of Formula I:
- R 1 is a 3'-0 reversible blocking group
- R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof
- R 3 consists of or comprises one or more phosphates; under conditions wherein the primer is extended to incorporate the unlabeled RT into a sequence complementary to at least some of said plurality of said template DNA molecules, thereby producing unlabeled extension products comprising the RT; (c) contacting the unlabeled extension products with an affinity reagent comprising a detectable label under conditions wherein the affinity reagent binds specifically to the RT to produce labeled extension products comprising the RT; and (d) identifying the RT in the labeled extension products to identify at least a portion of the sequence of said nucleic acid.
- such a method comprises: (b) contacting the DNA array with a nucleic acid primer complementary to a portion of each of said template DNA molecules, a polymerase, and a set of unlabeled RTs of Formula I that comprises a first RT in which R 2 is A, a second RT in which R 2 is T, a third RT in which R 2 is C, and a fourth RT in which R 2 is G, under conditions in which the primer is extended to incorporate the unlabeled RTs into sequences complementary to at least some of said plurality of said template DNA molecules, thereby producing unlabeled extension products comprising the RTs; (c) contacting the unlabeled extension products with a set of affinity reagents under conditions in which the set of affinity reagents binds specifically to the incorporated RTs to produce labeled extension products comprising the RTs, wherein: (i) the set of affinity reagents comprises a first affinity reagent that bind
- each of said first, second, third and fourth affinity reagents comprises a detectable label.
- each of said first, second, and third affinity reagents comprises a different detectable label.
- each of the first, second, and third affinity reagents comprises the same label (e.g., same fluorophore(s)) in different amounts, resulting in signals of different intensities.
- the affinity reagents bound to incorporated RTs are not directly labeled but are indirectly labeled using secondary affinity reagents.
- DNA arrays comprise: a plurality of template DNA molecules, each DNA molecule attached at a position of the array, a complementary DNA sequence base-paired with a portion of the template DNA molecule at a plurality of the positions, wherein the complementary DNA sequence comprises at its 3' end an incorporated RT; and an affinity reagent attached specifically to at least some of the RTs, the affinity reagent comprising a detectable label that identifies the RT to which it is attached.
- kits comprise: (a) an unlabeled RTs of Formula I:
- R 1 is a 3'-0 reversible blocking group
- R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof
- R 3 consists of or comprises one or more phosphates
- (c) packaging for the RT and the affinity reagent comprises: a plurality of the RTs, wherein each RT comprises a different nucleobase, and a plurality of affinity reagents, wherein each affinity reagent binds specifically to one of the RTs.
- Figure 1 is a flowchart that illustrates an example of a sequencing method of the invention.
- Figure 2 is a flowchart that illustrates an example of an antibody staining process shown in Figure 1.
- Figure 3 shows examples of NLRT structures: Fig. 3A 3'-0-azidomethyl-2'- deoxyguanine; Fig. 3B 3'-0-amino-2'-deoxyguanine; Fig. 3C 3'-0-cyanoethylene-2'- deoxyguanine; Fig. 3D 3'-0-phospho; Fig. 3E: 3'-ethyldisulfide-methylene-2'-deoxythymine.
- Figure 4 illustrates various blocking groups that can be used in the practice of the invention.
- Figure 4 indicates the attachment point of the molecule to the remainder of the structure.
- Figure 5 illustrates the synthesis of the active ester of 3'-0-azidomethyl-2'- deoxyguanine (G4).
- Figure 6 illustrates the synthesis of the active ester of 3'-0-azidomethyl-2'- (C8)
- Figure 7 illustrates the synthesis of the active ester of 3'-0-azidomethyl-2'- deoxyadenine (A12).
- Figure 8 illustrates the synthesis of the active ester of 3'-0-azidomethyl-2'- deoxythymine (T16).
- Figure 9 illustrates (using 3'-0-azidomethyl-2'-deoxycytosine) conjugation of 3'-
- Figures 10A and 10B show Rho for 5 and 10 cycles of sequencing using three labeled RTs and one NLRT.
- Figures IOC and 10D show Signal-Noise-Ratios (SNR) for 5 and 10 cycles of sequencing using three labeled RT and one NLRT.
- SNR Signal-Noise-Ratios
- Figures 11A and 11B illustrate sequencing data metrics obtained using the
- Figures 12A and 12B illustrate results from 25 sequencing cycles of E. coli genomic DNA on a BGISEQ-500 instrument using fluorescent directly labeled anti-azidomethyl- base antibodies.
- the present invention provides methods and compositions for sequencing-by-synthesis (SBS) or combinatorial probe anchor sequencing (cPAS) of nucleic acids that employ unlabeled reversible terminator nucleotides.
- SBS is carried out by producing immobilized single stranded template DNAs at positions on an array.
- each immobilized single stranded template DNA is at a position with a large number of copies (e.g., amplicons) of like sequence.
- bridge PCR may be used to generate a cluster of template sequences at a position on an array (lllumina), or rolling circle replication may be used to generate a single-stranded concatemer, or DNA nanoball (DNB), with many copies of the template sequences (Complete Genomics, Inc.).
- SBS is carried out by hybridizing a primer or primers to the template DNA and extending the primer to produce an extended primer, or growing DNA strand (GDS). Extending the primer refers to addition (“incorporation" or "incorporating") of nucleotides at the 3' end of the primer DNA strand while it is hybridized to the template.
- nucleotide incorporated at the 3' terminus is complementary to the corresponding nucleotide of the primer such that by determining the identity of the incorporated nucleotide at each sequencing cycle the nucleotide sequence of the template may be determined.
- labeled nucleotide analogs are incorporated into the
- the labeled nucleotide analogs comprise a blocking group that insures that only a single nucleotide per step can be incorporated and a dye (typically a fluorescent dye) attached via a cleavable linker to the nucleotide.
- a dye typically a fluorescent dye
- Each cycle of sequencing encompasses incorporating a labeled nucleotide analog at the end of the GDS, detecting the incorporated labeled nucleotide analog label, removing the label from the incorporated nucleotide analog, and removing the blocking group from the incorporated nucleotide analog to allow incorporation of a new labeled nucleotide analog.
- the present invention does not require labeled nucleotide analogs that include a dye attached via a cleavable linker to a base or sugar.
- a nucleotide analog when incorporated, comprises an affinity tag attached via a linker to the nucleotide.
- the affinity tag is one member of a specific binding pair (SBP).
- SBP specific binding pair
- the affinity tag is biotin.
- an affinity reagent comprising the second member of the SBP (e.g., streptavidin) and a detectable label.
- the detectable label is detected to identify the incorporated nucleotide.
- the incorporated nucleotide analog-affinity reagent complex is treated to cleave the linker and release the detectable label.
- the affinity tag is an antigen and the affinity reagent is a fluorescently labeled antibody that specifically binds the antigen.
- the present invention does not require an affinity tag and employs, in some aspects, an affinity reagent that binds the nucleobase, sugar moiety, cleavable blocking group or a combination thereof, rather than to an affinity tag.
- a non-labeled reversible terminator i.e., a nucleotide analog that includes a reversible terminator or blocking group (Non-Labeled Reversible Terminator, or NLRT)
- NLRT Non-Labeled Reversible Terminator
- an affinity reagent e.g., antibody
- nucleotide analog comprising a reversible blocking group is incorporated at the 3' terminus of the GDS, and after detection of the binding event, the reversible blocking group and the affinity reagent are removed, optionally in the same step.
- each cycle of sequencing includes: (i) incorporation of an NLRT comprising a blocking group by a DNA polymerase, followed by washing away unincorporated NLRT(s); (ii) contacting the incorporated nucleotide analog with an labeled affinity reagent that recognizes and specifically binds to the incorporated NLRT; (iii) detection of the binding of the affinity reagent; (iv) removal of the blocking group in a fashion that allows incorporation of an additional nucleotide analog (e.g., produces a hydroxy I group at the 3' position of a deoxyribose moiety), and (v) removal of the affinity reagent.
- an additional nucleotide analog e.g., produces a hydroxy I group at the 3' position of a deoxyribose moiety
- the affinity reagent e.g., antibody
- the affinity reagent may be directly labeled (e.g., a fluorescent labeled antibody) or may be detected indirectly (e.g., by binding of a labeled anti-affinity reagent secondary affinity reagent).
- a "labeled affinity reagent” may be directly labeled by, for example, conjugation to a fluorophore, or indirectly labeled.
- each cycle of sequencing includes: (i) incorporation of an NLRT comprising a blocking group by a DNA polymerase, optionally followed by washing away unincorporated NLRT(s); (ii) removal of the blocking group in a fashion that regenerates a hydroxy I (OH) group at the 3' positon of the deoxyribonucleotide; (iii) removing the blocking group of the allows incorporation of an additional nucleotide analog (e.g., produces a hydroxy I group at the 3' position of a deoxyribose moiety)contacting the incorporated nucleotide analog with an labeled affinity reagent that recognizes and specifically binds to the incorporated NLRT; (iii) detection of the binding of
- the affinity reagent e.g., antibody
- the affinity reagent may be directly labeled (e.g., a fluorescent labeled antibody) or may be detected indirectly (e.g., by binding of a labeled anti-affinity reagent secondary affinity reagent).
- a "labeled affinity reagent” may be directly labeled by, for example, conjugation to a fluorophore, or indirectly labeled.
- SBS involves two or more cycles of primer extension in which a nucleotide is incorporated at the 3' terminus of the extended primer.
- the present invention makes use of affinity reagents, such as antibodies, to (i) detect the nucleotide incorporated at the 3' terminus of the extended primer ("3' terminal nucleotide") and (ii) identify the nucleobase of that 3' terminal nucleotide and distinguishing one nucleobase from another (e.g., A from G).
- affinity reagents such as antibodies
- Each affinity reagent (or in some cases combination of affinity reagents) is also designed to detect properties of a 3' terminal nucleotide that identify the nucleobase associated with the 3' terminal nucleotide.
- a number of strategies, methods, and materials are provided for carrying out these and other steps. This section provides an overview in which many variations are omitted, and should not be considered limiting in any way.
- the SBS reactions of the invention are carried out using nucleotides with 3' reversible terminator moieties.
- the incorporated 3' terminal nucleotide differs from the internal nucleotides based on the presence of the reversible terminator moiety.
- an affinity reagent that binds to a reversible terminator moiety in an extended primer is binding to (and thereby detects) the 3' terminal nucleotide, distinguishing it from internal nucleotides.
- the incorporated 3' terminal nucleotide differs from the internal nucleotides based on the presence of a free 3' -OH (hydroxyl) group which is not present on internal nucleotides.
- an affinity reagent that binds to a free 3'-OH group in an extended primer is binding to the 3' terminal nucleotide is binding to (and thereby detects) the 3' terminal nucleotide, distinguishing it from internal nucleotides.
- the free 3'-OH group is generated by cleavage of the reversible terminator in an incorporated nucleotide analog.
- the free 3'-OH group results from incorporation of a nucleotide that does not comprise a reversible terminator moiety, such as a naturally occurring nucleotide.
- the incorporated 3' terminal nucleotide differs from the internal nucleotides based on other structural differences characteristic of a 3' terminal nucleotide including, but not limited to, greater accessibility of an affinity reagent to the deoxyribose sugar of a 3' terminal nucleotide relative to deoxyribose of internal nucleotides, greater accessibility of an affinity reagent to the nucleobase of a 3' terminal nucleotide to an affinity reagent relative to deoxyribose of internal nucleotides, and other molecular and conformational differences between the 3' terminal nucleotide and internal nucleosides.
- affinity reagents are used to detect these structural differences between the 3' terminal nucleotide of an extended primer and other nucleotides.
- naturally occurring nucleotides, or nucleotide analogs comprising naturally occurring nucleobases e.g., A, T, C and G
- Affinity reagents that specifically bind to one nucleobase (e.g., A) and distinguish that nucleobase from others to which it does not bind are used to identify the nucleobase of the 3' terminal nucleotide.
- nucleotide analogs comprising modified (i.e., not naturally occurring) nucleobases are used in the sequencing reaction and incorporated into the primer extension product.
- Affinity reagents that specifically bind to one modified nucleobase (e.g., modified A) and distinguish that modified nucleobase from other modified or natural nucleobases.
- An affinity reagent that specifically binds to a modified nucleobase generally recognizes the modification, such that the binding to modified nucleobase differs from binding to a naturally occurring nucleobase without the modification.
- an affinity reagent that binds to an adenosine analog in which nitrogen at position 7 (N 7 ) is replaced by methylated carbon may not bind to the naturally occurring (unmodified) adenosine nucleobase, or may bind less avidly.
- an affinity reagent that specifically recognizes a modified moiety in this case a modified nucleobase
- the affinity reagent binds an epitope that includes the methylated-carbon. It will be understood that the affinity reagent binds other portions of the incorporated nucleotide as well.
- nucleotides with 3' reversible blocking groups [0044]
- reversible terminator nucleotides are incorporated into the primer extension product.
- the blocking groups are removed at each sequencing cycle so that only the last incorporated nucleotide of the primer extension produce comprises a blocking group.
- affinity reagents that bind the blocking groups are used.
- at least two nucleotide analogs (i.e., with different nucleobases) used in the sequencing reaction comprise different blocking groups.
- a first blocking group e.g., 3'-0- azidomethyl
- a second, different blocking group e.g., 3'-0-cyanoethylene
- the specificity of the affinity reagent will identify the associated nucleobase. For example, extending the illustration above, if a 3' terminal nucleotide is recognized by an affinity reagent specific for 3'-0-cyanoethylene this indicates that the associated nucleobase is guanine or a guanine analog and the template base at this position is cytosine.
- blocking groups that differ by only a small feature may be used, and the affinity reagent binds an epitope that includes the distinguishing small feature.
- affinity reagents that recognize and specifically bind to nucleotides or nucleotide analogs based on a combination of structural features are used (e.g., an affinity reagent that recognizes a particular blocking group and a specific nucleobase with particular modifications) are used.
- nucleotides or nucleotide analogs are designed and/or selected for the property of being recognized by a specific affinity reagent.
- an affinity reagent that binds multiple structural features has the advantage of stronger and more specific affinity reagent binding.
- the portion of the incorporated nucleotide analog to which the labeled affinity reagent binds may include, for example and not limitation, the nucleobase and the blocking group, or the nucleobase and/or the blocking group in combination with the sugar moiety of the nucleotide analog. See Table A, below. Binding of the labeled affinity reagent may depend on the position of the target nucleotide, e.g., distinguishing between a nucleotide analog having a blocking group at the 3' terminus of the GDS, and a similar nucleotide analog (lacking the blocking group) that is located within or internal to the GDS.
- Binding of the labeled affinity reagent also depends upon the nucleobase itself, such that the affinity reagents binds to one target NLRT (e.g., NLRT-A) incorporated at the end of a GDS at one position on an array but not to other NLRTs (e.g., NLRT-C, -T, or -G) incorporated at the end of a GDS at a different position on an array.
- NLRT e.g., NLRT-A
- other NLRTs e.g., NLRT-C, -T, or -G
- the present invention has advantages over other SBS methods. Removal of the labeled affinity reagent does not leave behind a chemical "scar” resulting from groups left attached to the dNTP after cleavage of a linker. This is advantageous because such "scars” may reduce the efficiency of dNTP incorporation by polymerase.
- the affinity reagent may include multiple fluorescent moieties and provide a stronger signal than a single fluorescent dye attached to a dNTP according to commonly used methods. This approach also may cause less photodamage, since lower excitation power or shorter exposure times may be used.
- compositions and methods of the present invention also may be more economical than labeled reversible terminator (RT) methods commonly used for SBS.
- Unlabeled RTs cost less than labeled RTs. In standard SBS using labeled RTs, high concentrations of labeled RTs are used to drive the incorporation of the RT to completion, and most of the labeled RTs (70-99% or more) are not incorporated by polymerase and are washed away.
- a labeled affinity reagent in which a labeled affinity reagent is used, it may be sufficient for only a small percentage of target templates are bound by an affinity reagent that has multiple even 30% may be enough with efficiently labeled binders with multiple molecules of label to one molecule of binder; to be labeled (e.g., about 5%, or about 10%, or less than about 15%, less than about 20%, less than about 25%, or less than about 30%) in order to obtain a sufficient signal for imaging, particularly if the affinity reagent efficiently binds to the target dNTP and comprises multiple label molecules. A higher level of binding may be preferred if the affinity reagent bears only a single label molecule (e.g., 70 percent or more).
- nonlabled reversible terminator [nucleotide] As used herein, unless otherwise apparent from context, “nonlabled reversible terminator [nucleotide],” “NLRT,” “reversible terminator nucleotide,” “reversible terminator,” “RT,” and the like are all used to refer to a sequencing reagent comprising a nucleobase or analog, deoxyribose or analog, and a cleavable blocking group.
- a nonlabled reversible terminator nucleotide may refer to a dNTP (i.e., a substrate for polymerase) or a reversible terminator nucleotide incorporated to into a primer extension product, initially at the 3' terminus and, following additional incorporation cycles, if any, in an "internal" portion of the primer extension product.
- dNTP i.e., a substrate for polymerase
- a "dNTP” includes both naturally occurring deoxyribonucleotide triphosphates and analogs thereof, including analogs with a 3'-0 cleavable blocking group.
- Amplicons may be produced by a variety of amplification reactions, including but not limited to polymerase chain reactions (PCRs), linear polymerase reactions, nucleic acid sequence-based amplification, rolling circle amplification and like reactions (see, e.g., U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159; 5,210,015; 6,174,670; 5,399,491; 6,287,824 and 5,854,033; and U.S. Pub. No. 2006/0024711).
- PCRs polymerase chain reactions
- Antigen as used herein means a compound that can be specifically bound by an antibody. Some antigens are immunogens (see, Janeway, et al., Immunobiology, 5th Edition, 2001, Garland Publishing). Some antigens are haptens that are recognized by an antibody but which do not elicit an immune response unless conjugated to a protein. Exemplary antigens include NLRTs, reversible terminator blocking groups, dNTPs, polypeptides, small molecules, lipids, or nucleic acids.
- Array or “microarray” means a solid support (or collection of solid supports such as beads) having a surface, preferably but not exclusively a planar or substantially planar surface, which carries a collection of sites comprising nucleic acids such that each site of the collection is spatially defined and not overlapping with other sites of the array; that is, the sites are spatially discrete.
- the array or microarray can also comprise a non-planar interrogatable structure with a surface such as a bead or a well.
- the oligonucleotides or polynucleotides of the array may be covalently bound to the solid support, or it may be non-covalently bound.
- random array or “random microarray” refers to a microarray where the identity of the oligonucleotides or polynucleotides is not discernable, at least initially, from their location but may be determined by a particular biochemistry detection technique on the array. See, e.g., U.S. Pat. Nos. 6,396,995; 6,544,732; 6,401,267; and 7,070,927; PCT publications WO 2006/073504 and 2005/082098; and U.S. Pat. Pub. Nos. 2007/0207482 and 2007/0087362.
- reversible in reference to a blocking group have the same meaning.
- reversible blocking group of a reversible terminator nucleotide may also be referred to as a “removable blocking group,” a “cleavable linker,” a “blocking moiety,” a “blocking group,” “reversible terminator blocking group” and the like.
- a reversible blocking group is a chemical moiety attached to the nucleotide sugar (e.g., deoxyribose), usually at the 3' -O position of the sugar moiety, which prevents addition of a nucleotide by a polymerase at that position.
- a reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), chemical reaction, heat, light, etc., to provide a hydroxy I group at the 3'-position of the nucleoside or nucleotide such that addition of a nucleotide by a polymerase may occur.
- an enzyme e.g., a phosphatase or esterase
- chemical reaction e.g., heat, light, etc.
- “Derivative” or “analogue” means a compound or molecule whose core structure is the same as, or closely resembles that of, a parent compound, but which has a chemical or physical modification, such as a different or additional side group, or 2' and or 3' blocking groups, which allows the derivative nucleotide or nucleoside to be linked to another molecule.
- the base can be a deazapurine.
- the derivatives should be capable of undergoing Watson-Crick pairing.
- “Derivative” and “analogue” also mean a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties.
- Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate and phosphoramidate linkages.
- the analogs should be capable of undergoing Watson-Crick base pairing.
- deoxyadenosine analogues include didanosine (ddl) and vidarabine, and adenosine analogues include, BCX4430; deoxycytidine analogs include cytarabine, gemcitabine, emtricitabine (FTC), lamivudine (3TC), and zalcitabine (ddC); guanosine and deoxyguanosine analogues include abacavir, aciclovir, and entecavir; thymidine and deoxythymidine analogues include stavudine (d4T), telbivudine, and zidovudine (azidothymidine, or AZT); and deoxyuridine analogues include idoxuridine and trifluridine.
- deoxycytidine analogs include cytarabine, gemcitabine, emtricitabine (FTC), lamivudine (3TC), and
- incorporation of an RT occurs when a polymerase adds an RT to a growing DNA strand through the formation of a phosphodiester or modified phosphodiester bond between the 3' position of the pentose of one nucleotide, that is, the 3' nucleotide on the DNA strand, and the 5' position of the pentose on an adjacent nucleotide, that is, the RT being added to the DNA strand.
- Label in the context of a labeled affinity reagent, means any atom or molecule that can be used to provide a detectable and/or quantifiable signal. Suitable labels include radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.
- the detection label is a molecule containing a charged group (e.g., a molecule containing a cationic group or a molecule containing an anionic group), a fluorescent molecule (e.g., a fluorescent dye), a fluorogenic molecule, or a metal.
- the detection label is a fluorogenic label.
- a fluorogenic label can be any label that is capable of emitting light when in an unquenched form (e.g., when not quenched by another agent).
- the fluorescent moiety emits light energy (i.e., fluoresces) at a specific emission wavelength when excited by an appropriate excitation wavelength. When the fluorescent moiety and a quencher moiety are in close proximity, light energy emitted by the fluorescent moiety is absorbed by the quencher moiety.
- the fluorogenic dye is a fluorescein, a rhodamine, a phenoxazine, an acridine, a coumarin, or a derivative thereof.
- the fluorogenic dye is a carboxyfluorescein.
- suitable fluorogenic dyes include the fluorogenic dyes commercially available under the Alexa Fluor" product line (Life Technologies, Carlsbad, CA).
- non-fluorogenic labels may be used, including without limitation, redoxgenic labels, reduction tags, thio- or thiol-containing molecules, substituted or unsubstituted alkyls, fluorescent proteins, non-fluorescent dyes, and luminescent proteins.
- Nucleobase means a nitrogenous base that can base-pair with a complementary nitrogenous base of a template nucleic acid.
- Exemplary nucleobases include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), inosine (I) and derivatives of these. References to thymine herein should be understood to refer equally to uracil unless otherwise clear from context.
- the terms "nucleobase,” “nitrogenous base,” add “base” are used interchangeably.
- a "naturally occurring nucleobase,” as used herein, means adenine (A), cytosine
- nucleobase refers to A, C, G and T (the naturally occurring bases found in DNA).
- a "nucleotide” consists of a nucleobase, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence.
- the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxy I group that is present in ribose.
- the nitrogenous base is a derivative of purine or pyrimidine.
- the purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) (or in the context of RNA, uracil (U)).
- the C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine.
- a nucleotide is also a phosphate ester or a nucleoside, with esterification occurring on the hydroxy I group attached to C-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.
- a "nucleoside” is structurally similar to a nucleotide, but does not include the phosphate moieties. Common abbreviations include "dNTP" for deoxy nucleotide triphosphate.
- Nucleic acid means a polymer of nucleotide monomers. As used herein, the terms may refer to single- or double-stranded forms. Monomers making up nucleic acids and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like, to form duplex or triplex forms. Such monomers and their internucleosidic linkages may be naturally occurring or may be analogs thereof, e.g., naturally occurring or non-naturally occurring analogs.
- Non-naturally occurring analogs may include peptide nucleic acids, locked nucleic acids, phosphorothioate internucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like.
- Nucleic acids typically range in size from a few monomeric units, e.g., 5-40, when they are usually referred to as "oligonucleotides,” to several hundred thousand or more monomeric units.
- nucleic acid or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG,” it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, "U” denotes uridine, unless otherwise indicated or obvious from context. Unless otherwise noted the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999).
- nucleic acids comprise the natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g., modified bases, sugars, or internucleosidic linkages.
- natural nucleosides e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA
- non-natural nucleotide analogs e.g., modified bases, sugars, or internucleosidic linkages.
- an enzyme has specific oligonucleotide or nucleic acid substrate requirements for activity, e.g., single-stranded DNA, RNA/DNA duplex, or the like
- selection of appropriate composition for the oligonucleotide or nucleic acid substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references.
- Primer means an oligonucleotide, either natural or synthetic, which is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed.
- the sequence of nucleotides added during the extension process are determined by the sequence of the template polynucleotide.
- primers are extended by a DNA polymerase. Primers usually have a length in the range of from 9 to 40 nucleotides, or in some embodiments, from 14 to 36 nucleotides.
- DNA, RNA, and hybrid and synthetic nucleic acids may be single-stranded or double- stranded.
- Olionucleotides are short polynucleotides of between about 6 and about 300 nucleotides in length.
- “Complementary polynucleotide” refers to a polynucleotide complementary to a target nucleic acid.
- Solid support and “support” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces.
- Microarrays usually comprise at least one planar solid phase support, such as a glass microscope slide.
- SBS according to the invention may use non-labeled reversible terminators ("NLRT”) (e.g., a nucleotide analog with a blocking group), non-labeled naturally occuring nucleotides (e.g., dATP, dTTP, dCTP and dGTP), or non-labeled nucleotide analogs that do not include a blocking group.
- NLRT non-labeled reversible terminators
- Non-labeled reversible terminators are nucleotide analogs comprising a removable blocking group at the 3'-OH position of the deoxyribose. Although numerous reversible terminators have been described, and reversible terminators are widely used in SBS, the non-labeled reversible terminators used in accord with the present invention differ from those in commercial use because they are non-labeled and because they are used in conjunction with the affinity reagents described herein below.
- the NLRTs of the invention are non-labeled.
- non-labeled means the NLRT does not comprise a fluorescent dye.
- non-labeled means the NLRT does not comprise a chemiluminescent dye.
- non-labeled means the NLRT does not comprise a light emitting moiety.
- exemplary NLRTs have Structure I, below, prior to incorporation of the NLRT into a DNA strand.
- R 1 is a 3'-0 reversible blocking group
- R 2 is, or includes, the nucleobase
- R 3 comprises at least one phosphate group or analog thereof.
- Reversible blocking groups R 1 may be removed after incorporation of the NLRT into a DNA strand. After incorporation of the analog at the 3' terminus of a DNA strand, the removal of the blocking group results in a 3'-OH. Any reversible blocking group may be used. Exemplary reversible blocking groups are described below.
- Nucleobases R 2 may be, for example, adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or inosine (I) or analogs thereof.
- NLRTs may be referred to according to the nucleobase; for example, an NLRT that has an A nucleobase is referred to as NLRT-A.
- the corresponding NLRTs are referred to herein as "NLRT-A,” “NLRT-C,” “NLRT-G,” “NLRT-T,” “NLRT-U,” and “NLRT-I,” respectively.
- NLRT-T and NLRT-C may be referred to as NLRT- pyrimidines.
- NLRT-G and NLRT-A may be referred to as NLRT-purines.
- Nucleobase R 2 may be any nucleobase or nucleobase analog (e.g., an analog of adenine, cytosine, guanine, thymine, uracil, or inosine). For example, a modification to the naturally occurring nucleobase may be made to increase the immune response to the analog when raising antibodies, or to increase the specificity of the antibody(s) for specific nucleobase.
- R 3 may be 1-10 phosphate or phosphate analog groups.
- Phosphate analogs include phosphorothioate (PS), in which the phosphorothioate bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of the DNA, or any other suitable phosphate analog known in the art.
- R 3 may be 1-10 phosphate groups. In some cases, R 3 may be 3-12 phosphate groups.
- the nucleotide analogue is a nucleoside triphosphate.
- R 1 of Formula I has a MW less than 184, often less than
- R 1 may act as a hapten and elicit an immune response when conjugated to a larger carrier molecule such as KLH.
- the unincorporated NLRT nucleotide analogue is suitable as a substrate for an enzyme with DNA polymerase activity and can be incorporated into a DNA strand at the 3' terminus.
- the reversible blocking group should have a size and structure such that the NLRT is a substrate for at least some DNA polymerases.
- the incorporation of an NLRT may be accomplished via a terminal transferase, a polymerase or a reverse transcriptase.
- Any DNA polymerase used in sequencing may be employed, including, for example, a DNA polymerase from Thermococcus sp., such as 9" N or mutants thereof, including A485L, including double mutant Y409V and A485L.
- polymerases are highly discriminating with regard to the nature of the 3' blocking group. As a result, mutations to the polymerase protein are often needed to drive efficient incorporation.
- Exemplary DNA polymerases and methods that may be used in the invention include those described in Chen, C, 2014, "DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present" Frontiers in Microbiology, Vol. 5, Article 305, Pinheiro, V. et al.
- the polymerase is DNA polymerase from Thermococcus sp., such as 9° N or mutants thereof, including A485L, including double mutant Y409V and A485L.
- Other examples include £ coli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, HIV reverse transcriptase; Phi29 polymerase, and Bst DNA polymerase.
- the RTs have Structure II, below, prior to incorporation of the
- R 1 is a 3'-0 reversible blocking group
- R 4 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U); and R 3 comprises at least one (e.g., 1-10) phosphate. In some cases, R 3 IS triphosphate.
- the RTs have Structure III, below, after incorporation of the RT into a DNA strand.
- R 1 is a 3'-0 reversible blocking group
- R 2 is a nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or inosine (I) or analogs thereof
- X is a polynucleotide (e.g., GDS) comprising 10-1000 nucleosides linked by phosphate-sugar bonds (e.g., phosphodiester bonds linking the 3' carbon atom of one nucleoside sugar molecule and the 5' carbon atom of another nucleoside sugar molecule).
- the RTs have Structure IV, after incorporation and removal of the reversible blocking group.
- Re is H and R7 is a polynucleotide (e.g., GDS) comprising 10-1000 nucleosides linked by phosphate-sugar bonds, as defined above, or is R 3 , as defined above.
- GDS polynucleotide
- R 2 is a nucleobase analog (e.g., an analog of A, T, G, C, U) with modifications that do not change the binding specificity of the base (i.e., A analog binds T, T analog binds A, etc.) and (ii) but which may render the analog more immunogenic than the naturally occurring base.
- the modification may comprise additions of a group comprising no more than 3 carbons. The added group is not removed from nucleosides as they are incorporated into the GDS so that the GDS comprises a plurality of nucleotides comprising the modification.
- the affinity reagent binds the terminal nucleotide analog, including the modification, but binds internal nucleotides with the modification with much lower affinity.
- An NLRT used in the present invention can include any suitable blocking group.
- a suitable blocking group is one that may be removed by a chemical or enzymatic treatment to produce a 3'-OH group.
- a chemical treatment should not significantly degrade the template or primer extension strand.
- 3' blocking group of reversible terminators such as a 3'-0-allyl group (Ju et al., Proc. Natl. Acad. Sci. USA 103: 19635-19640, 2006), 3'-0-azidomethyl-dNTPs (Guo et al., Proc. Natl Acad. Sci.
- RT blocking groups include -O-azidomethyl and -O-cyanoethenyl.
- Other exemplary RT blocking groups are shown in FIGURES 3 and 4.
- R 1 of Formula I is a substituted or unsubstituted a Iky I, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, or substituted or unsubstituted heteroalkynyl.
- R 1 can be selected from the group consisting of allenyl, cis-cyanoethenyl, trans-cyanoethenyl, cis-cyanofluoroethenyl, trans- cyanofluoroethenyl, cis-trifluoromethylethenyl, trans-trifluoromethylethenyl, biscyanoethenyl, bisfluoroethenyl, cis-propenyl, trans-propenyl, nitroethenyl, acetoethenyl, methylcarbonoethenyl, amidoethenyl, methylsulfonoethenyl, methylsulfonoethyl, formimidate, form hydroxy mate, vinyloethenyl, ethylenoethenyl, cyanoethylenyl, nitroethylenyl, amidoethylenylenyl,
- R 1 is selected from the group consisting of ally I, azidomethyl, aminoalkoxyl, 2-cyanoethyl, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heteroalkenyl, unsubstituted heteroalkenyl, substituted heteroalkynyl, unsubstituted heteroalkynyl, allenyl, cis-cyanoethenyl, trans-cyanoethenyl, cis-cyanofluoroethenyl, trans- cyanofluoroethenyl, cis-trifluoro
- a Iky I straight- and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. Ranges of these groups useful with the compounds and methods described herein include Ci-Cio alkyl, C2-C10 alkenyl, and C2-C10 alkynyl.
- Ci-Q alkyl C2-C8 alkenyl, C2-C8 alkynyl, Ci-C 6 alkyl, C 2 -C 6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.
- Heteroalkyl “heteroalkenyl,” and “heteroalkynyl” are defined similarly as alkyl, alkenyl, and alkynyl, but can contain O, S, or N heteroatoms or combinations thereof within the backbone. Ranges of these groups useful with the compounds and methods described herein include C1-C10 heteroalkyl, C2-C10 heteroalkenyl, and C2-C10 heteroalkynyl.
- Ci-Ce heteroalkyl C 2 -Ce heteroalkenyl, C 2 -Cs heteroalkynyl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, C 2 - Q heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.
- alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl molecules used herein can be substituted or unsubstituted.
- substituted includes the addition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to a position attached to the main chain of the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl, e.g., the replacement of a hydrogen by one of these molecules.
- substitution groups include, but are not limited to, hydroxy, halogen (e.g., F, Br, CI, or I), and car boxy I groups.
- halogen e.g., F, Br, CI, or I
- car boxy I groups e.g., hydroxy, halogen (e.g., F, Br, CI, or I)
- the term unsubstituted indicates the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl has a full complement of hydrogens, i.e., commensurate with its saturation level, with no substitutions, e.g., linear butane (-(CH2)r-CH3).
- the reversible blocking group is an amino-containing blocking group (e.g., NHr-)- See, H utter et al., 2010, Nucleosides Nucleotides Nucleic Acids 29(11), incorporated herein by reference, which describes exemplary amino-containing reversible blocking groups.
- the reversible blocking group comprises a cyano group (e.g. a cyanoethenyl or cyanoethyl group).
- the reversible blocking group is an azido-containing blocking group (e.g., Nr-). In some embodiments, the reversible blocking group is azidomethyl (N3CH2-). In some embodiments, the reversible blocking group is an alkoxy-containing blocking group (e.g., CH3CH2O-). In some embodiments, the reversible blocking group contains a polyethylene glycol (PEG) moiety with one or more ethylene glycol units. In some embodiments, the reversible blocking group is a substituted or unsubstituted a Iky I (i.e., a substituted or unsubstituted hydrocarbon). In some embodiments, the reversible blocking group is acyl.
- the reversible blocking group is nitrobenzyl (Ceh f C ⁇ )-
- the reversible blocking group is coumarinyl (i.e., contains a coumarin moiety or a derivative thereof) wherein, e.g., any one of the CH carbons of the coumarinyl reversible blocking group is covalently attached to the 3'-0 of the nucleotide analogue.
- the reversible blocking group is nitronaphthalenyl (i.e., contains a nitronaphthalene moiety or a derivative thereof) wherein, e.g., any one of the CH carbons of the nitronaphthalenyl reversible blocking group is covalently attached to the 3'-0 of the nucleoside analogue.
- the reversible blocking group is selected from the group:
- R 3 and R 4 are H or a Iky I
- R5 is a Iky I, cycloalkyl, alkenyl, cycloalkenyl, and benzyl.
- the determination of R 3 -R5 is constrained by MW limitations described herein (e.g., see Section 3.2.1).
- reversible blocking groups suitable for use in the present invention are described in the literature as a blocking group of a labeled reversible terminator. Generally any suitable reversible blocking group used in sequencing-by-synthesis may be used in the practice of the invention.
- the blocking group of RTs is removable under reaction conditions that do not interfere with the integrity of the DNA being sequenced.
- the ideal blocking group will exhibit long term stability, be efficiently incorporated by the polymerase enzyme, cause total blocking of secondary or further incorporation and have the ability to be removed under mild conditions that do not cause damage to the polynucleotide structure, preferably under aqueous conditions.
- a blocking group (including the deoxyribose 3' oxygen atom) has a molecular weight (MW) less than 200, often less than 190, often less than 180, often less than 170, often less than 160, often less than 150, often less than 140, often less than 130, often less than 120, often less than 110, and sometimes less than 100).
- R 3 of Formula I has a MW less than 184, often less than 174, often less than 164, often less than 154, often less than 144, often less than 134, often less than 124, often less than 114, often less than 104, often less than 94, and sometimes less than 84.
- the molecular weights of deoxyribonucleotide monophosphates are in the range of about 307 to 322 (dAMP 331.2, dCMP 307.2, dGMP 347.2 and dTMP 322.2).
- the NLRT moiety when incorporated into a GDS has a molecular weight less than 550, often less than 540, often less than 530, often less than 520, often less than 510, often less than 500, often less than 490, often less than 480, often less than 470, and sometimes less than 460.
- the R 3 moiety comprises one or more phosphate and/or phosphate analog moieties.
- X may be alkyl or any of a variety of linkers described in the art. See, e.g., U.S. Pat. No. 9,702,001, incorporated herein by reference.
- moiety X is removed from the nucleotide (along with all but the alpha phosphate) such that X is not present in the incorporated reversible terminator deoxyribonucleotide.
- X may be a detectable label or affinity tag, with the proviso that affinity reagents of the invention do not bind to moiety X, or discriminate among, reversible terminators based on the presence, absence or structure of moiety X, and that X is not present in the incorporated reversible terminator deoxyribonucleotide.
- SBS sequencing according to the invention comprises contacting a sequencing array with multiple NLRTs (e.g., NLRT- A, NLRT-T, NLRT-C and NLRT-G).
- the contacting may be carried out sequentially, one NLRT at a time.
- the four NLRTs may be contacted with the sequencing array at the same time, most often as a mixture of the four NLRTs.
- the four NLRTs make up an "NLRT set.”
- NLRTs of an NLRT set may be packaged as a mixture or may be packaged as a kit comprising each different NLRT is a separate container. In a mixture of the four NLRTs may include each base in equal proportion or may include unequal amounts.
- each NLRT in an NLRT set comprises the same blocking group (e.g. azidomethyl).
- NLRTs in an NLRT set comprise different blocking groups (e.g. NLRT-A comprises azidomethyl and NLRT-T comprises cyanoethenyl; or NLRT-A and NLRT-G comprise azidomethyl and NLRT-C and NLRT-T comprise cyanoethenyl).
- different blocking groups are used, such blocking groups are optionally selected such that the different blocking group can be removed by the same treatment. Alternatively the blocking groups may be selected to be removed by different treatments, optionally at different times.
- one or more NLRTs in a set comprises a modified (nonnaturally occurring nucleobase).
- NLRTs described herein can be provided or used in the form of a mixture.
- the mixture can contain two, three, or four (or more) structurally different NLRTs.
- the structurally different NLRTs can differ in their respective nucleobases.
- the mixture can contain four structurally different NLRTs each comprising one of the four natural DNA nucleobases (i.e., adenine, cytosine, guanine, and thymine), or derivatives thereof.
- kits comprising NLRT sets (with different NLRTs packaged in separate containers or as a mixture in the same container) may be provided.
- the nucleobase includes a non-removable chemical group that increases the specificity or affinity of the affinity reagent for the nucleobase when present at the 3' terminus of the growing DNA strand (i.e., as the last-incorporated base), but which is not recognized by, or not accessible to, the affinity reagent in nucleotides internal to the primer extension product.
- the modification is recognized by or bound by the affinity reagent but with a lower affinity or lower efficiency relative to the same modification in a 3' terminal nucleotide.
- modified nucleobases include:
- Re, R7, Re, and R9 may be the same or different, each selected from H, I, Br, F, Structures XIX- XXVIII, or any groups that do not interfere with base pairing. Note that when R9 is methyl Structure XVIII in thymidine. In some cases, the modification has the additional benefit of increasing the antigenicity of the nucleotide.
- nucleobases are: adenine 135; guanine 151, thymine 126 and cytosine 111.
- nucleobase analog has a molecular weight that does not exceed that of the natural base by more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 Da.
- natural dNTPs e.g., dATP, dGTP, dCTP or dTTP
- dNTP analogs without a 3'-0- blocking group are used for sequencing.
- the nucleotides are incorporated one at a time in the sequencing process, as in pyrosequencing or by a polymerase that halts after one base incorporation. Exemplary methods are described in the literature (see, e.g., Ju et al., 2006, Proc. Natl. Acad. Sci. USA 103:19635-40, 2006; Guo, Proc. Natl Acad. Sci.
- dNTPs with different nucleobases are added and incorporated sequentially (e.g., A, then G, etc.). Usually nucleobase is separately imaged prior to addition of the next dNTP.
- the sugar (deoxyribose) moiety is modified.
- an NLRT with the nucleobase adenine, the blocking group azidomethyl, and the sugar deoxyribose can be distinguished from an NLRT with the nucleobase cytosine, the blocking group azidomethyl, and the sugar modified-deoxyribose using an affinity reagent that so that it is recognizes the blocking group and sugar moieties.
- a nucleotide with a nonremovable (i.e., not cleavable) 3' blocking group is used in place of a NLRT.
- the last-incorporated base is removed and its position is filed in with a nucleotide that is similar but that has a cleavable blocking group (Koziolkiewicz et al., FEBS Lett. 434:77-82, 1998).
- the examples given above include reversible blocking groups attached to the nucleotide via the 3'-0 of the deoxyribose sugar moiety.
- the present invention also includes NLRTs with reversible and non-reversible blocking groups attached to the 2'-0- of the deoxyribose sugar. These embodiments may be used for single base detection (single or a few base primer extension), monitoring gaps and nicks in DNA and other detection methods. Thus, one of ordinary skill in the art will be able to apply the methods and information herein to NLRTs with 2', rather than 3', blocking groups.
- the present invention uses affinity reagents that specifically bind to NLRTs at the
- the affinity reagent binds an NLRT of Structure III. In one embodiment the affinity reagent binds an NLRT of Structure IV.
- an affinity reagent used to detect the presence or absence of an NLRT incorporated at the 3' end of a nucleic acid.
- An affinity reagent is a molecule or macromolecule that specifically binds an NLRT based on a structural feature of the incorporated NLRT.
- an affinity reagent may specifically bind to an NLRT having, e.g., a particular base and/or particular reversible blocking group.
- an affinity reagent is a monoclonal antibody (mAb) that binds with high affinity to an incorporated NLRT at the 3' end of a DNA strand when the NLRT comprises the nucleobase adenosine and an azidomethyl reversible blocking group but does not bind with high affinity to an NLRT incorporated at the 3' end of a DNA strand when the NLRT comprises the nucleobase adenosine but has a 3' hydroxy I group rather than an azidomethyl reversible blocker, and does not bind with high affinity to an NLRT incorporated at the 3' terminus of a DNA strand comprising the nucleobase cytosine, guanine, or thymine, each with or without an azidomethyl reversible blocking group.
- Affinity reagents may be directly or indirectly labeled.
- Specificity is the degree to the affinity reagent discriminates between different molecules (e.g., NLRTs) as measured, for example, by relative binding affinities of the affinity reagent for the molecules.
- an affinity reagent should have substantially higher affinity for one NLRT (its target RT) than for other NLRTs (for example, the affinity reagent binds to a C nucleoside analogue but not to A, T or G).
- the affinity reagent binds to its target nucleoside analog at the end of a polynucleotide when incorporated by a polymerase at the 3' end of a growing DNA chain, but not to a nucleotide base elsewhere on the DNA chain.
- An affinity reagent is specific for a particular NLRT, such as NLRT-A, if in the presence of a plurality (e.g., an array) of template polynucleotides are present in which 3' -termini of GDSs include NLRT-A, NLRT-T, NLRT-C, NLRT- G (e.g., in an array) the affinity reagent binds preferentially to NLRT-A under reaction conditions used in SBS sequencing.
- affinity binding of an affinity agent to a first structure compared to a second structure means the affinity agent binds the first structure but does not bind the second structure or binds the second structure less strongly (i.e., with a lower affinity) or less efficiently.
- the terms “specific binding,” “specifically binds,” and the like refer to the preferential association of an affinity reagent with a particular NLRT (e.g., NLRT-A having a 3'-0 azido group) in comparison to an NLRT with a different nucleobase (NLRT-T, -C, or -G), a different blocking group, or no blocking group (e.g., deoxyadenosine with a 3' -OH).
- Specific binding between an affinity reagent and the NLRT sometimes means an affinity of at least 10 -6 NT 1 (i.e., an affinity having a lower numerical value than lO -6 M -1 as measured by the dissociation constant Kd). Affinities greater than 10 -8 M -1 are preferred. Specific binding can be determined using any assay for binding (e.g., antibody binding) known in the art, including Western Blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunohistochemistry, and detection of fluorescently labeled affinity reagent bound to a target NLRT in a sequencing reaction. As discussed herein below, specificity of binding can be determined by positive and negative binding assays.
- an affinity reagent such as an antibody
- an incorporated reversible terminator deoxyribonucleotide can be described in various ways including with reference to the portion, or moiety, of the incorporated reversible terminator deoxyribonucleotide responsible for the specificity.
- An analogy is useful here: Imagine a protein with two domains, domain 1 and domain 2. Two different antibodies may specifically bind the protein. However, they may recognize different epitopes. For example, one antibody may bind an epitope in domain 1 and the second antibody may bind an epitope in domain 2. In this hypothetical, if modifications are made in domain 1 this may affect the binding of the protein by the first antibody, without changing the binding by the second antibody.
- domain 1 may be said to be "responsible for" binding by antibody 1.
- domain 1 may be said to be due to a structural feature of one moiety (e.g., the blocking group) and be unaffected by the structure of other moieties (e.g., the nucleobase) by other moieties.
- binding by an affinity reagent to an incorporated reversible terminator deoxyribonucleotide requires the presence of particular structural features of a moiety
- the binding by the affinity reagent may "be specific for” or “based on” the presence or absence of a moiety with those structural features.
- the moiety with those structural features may be "responsible” for binding by the affinity reagent, or binding of the affinity reagent may be “dependent” on the presence of a moiety with those structural features.
- affinity may depend on the environment. For example, imagine an affinity reagent that binds both A and A', but does not bind B, C or D. In a reaction or sample containing A, A', B and C, the affinity reagent may bind both A and A', and thus may not be considered to "specifically bind" A. However, in a reaction or sample containing A, B, C and D, the affinity reagent would bind only A, and in that environment would be said to specifically bind A. In another example, in a sample containing A, A', B and C, the affinity reagent may bind A and A' with different affinities, or efficiencies, so that the binding to A and the binding to A' could be distinguished on those bases.
- Another related term is "discriminate” (or sometimes “distinguish”).
- blocking group e.g., azidomethyl
- an affinity reagent is a result of the process used to make the affinity reagent.
- a reagent that recognizes an azidomethyl blocking moiety may be tested empirically with positive and negative binding assays.
- the reagent is an antibody that binds an NLRT based on the presence of an O-azidomethyl blocking moiety.
- antibodies are raised against the hapten O-azidomethyl using azidomethyl conjugated to keyhole limpet hemocyanin.
- the desired antibody can be selected for binding to 3'-0-azidomethyl-2'-deoxyguanine but against binding to other deoxyguanine nucleotides such as 3'-0-2-(cyanoethoxy)methyl-2'-deoxyguanine; 3'-0-(2-nitrobenzyl)-2'- deoxyguanine; and 3'-0-allyl-2'-deoxyguanine; and against binding other azidomethyl NLRTs such as 3'-0-azidomethyl-2'-deoxyadenosine; 3'-0-azidomethyl-2'-deoxycytosine; and 3'-0- azidomethyl-2'-deoxythymine.
- deoxyguanine nucleotides such as 3'-0-2-(cyanoethoxy)methyl-2'-deoxyguanine; 3'-0-(2-nitrobenzyl)-2'- deoxyguanine; and 3'-0-allyl-2'-deoxyguanine
- azidomethyl NLRTs such as
- an affinity reagent as binding certain moieties (e.g., a nucleobase and a sugar moiety) does not exclude binding to other parts of the incorporated nucleotide.
- an affinity reagent that binding a nucleobase and a sugar moiety may also bind a blocking group.
- affinity reagents include antibodies (including binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like), aptamers, knottins, affimers, labeled dNTPs that form a one-base triple helix, guanine nucleotide binding proteins (G-proteins), or any other known agent that binds an incorporated NLRT with a suitable specificity and affinity.
- antibodies including binding fragments of antibodies, single chain antibodies, bispecific antibodies, and the like
- aptamers include knottins, affimers, labeled dNTPs that form a one-base triple helix, guanine nucleotide binding proteins (G-proteins), or any other known agent that binds an incorporated NLRT with a suitable specificity and affinity.
- G-proteins guanine nucleotide binding proteins
- the affinity reagent may specifically recognize the nucleobase, the sugar (e.g., deoxyribose), the blocking group, or any other moiety or combination thereof in the target NLRT.
- the affinity reagent recognizes an epitope comprising the blocking group.
- the affinity reagent recognizes an epitope comprising the nucleobase.
- the affinity reagent recognizes an epitope comprising the nucleobase and the blocking group. It will be understood that even if the affinity reagent does not contact a moiety, the moiety may dictate the position of other moieties.
- the deoxyribose moiety is required to position a nucleobase and 3' blocking group for recognition.
- affinity reagents that are antibodies
- specific binding can be determined using any assay for antibody binding known in the art, including Western Blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, or column chromatography.
- ELISA enzyme-linked immunosorbent assay
- specific binding is demonstrated using an ELISA type assay.
- serum antibodies raised against 3'-azidomethyl-dC can be serially titrated against a bound substrate of 3'-0-azidomethyl-dC (positive specificity assay) and nucleotide(s) such as 3'-0-azidomethyl-dG or -dA or 3'-OH-dC (negative specificity assay).
- the base-specific binding of an affinity reagent for its target nucleoside is 2-100-fold higher than binding to other nucleosides or analogs. In some embodiments base-specific binding of an affinity reagent for its target nucleoside is at least 10- fold higher than binding to other nucleosides, or at least 30-fold higher, or at least 100-fold higher
- the preferred the antibody binding efficiency to the specific base is at the concentration lower than 100 pM, or lower than 1 nM, or lower than 10 nM, or lower than ⁇ .
- affinity reagents with desired specificity can be selected using art-known methods. For example, an affinity reagent such as an antibody can be identified, selected, or purified by rounds of positive selection (i.e., binds to target molecule) and negative selection (i.e., does not binds to molecules that are not target molecule).
- An affinity reagent may bind both a dNTP in solution and the corresponding nucleotide incorporated at the 3' terminus of a primer extension product.
- the affinity reagent does not bind an unincorporated NLRT (e.g., an NLRT in solution) or binds with a significantly lower specificity.
- binding of non- incorporated NLRTs by affinity reagents does not occur in the process of sequencing because unincorporated NLRTs are removed (washed away) prior to introduction of the affinity reagents.
- complexes formed by affinity reagents bound to NLRTs are removed (washed away) prior to imaging.
- the affinity reagent binds specifically to the nucleobase and distinguishes among different bases (e.g., A, T, G, C) in part based on the presence or absence of a 3'-OH group.
- the affinity reagent distinguishes a nucleotide at the 3' end of a GDS with a 3'-OH from incorporated nucleotides interior to the GDS (not at the 3' end).
- the affinity reagent that recognizes a specific nucleobase also distinguishes between the presence or absence of a 3'-OH groups, thereby recognizing an incorporated NLRT as a 3' terminal nucleotide with a particular nucleobase.
- affinity reagent recognizes an epitope comprising the blocking group but does not distinguish between bases.
- affinity reagents can be produced that distinguish the four blocking groups.
- an affinity reagent can be selected that recognizes only one, but not the other three, NLRTs.
- the selected affinity reagent does not distinguish between nucleotides with different nucleobases provided they share the same blocking group.
- an affinity reagent that recognizes B (3'-0--2-(cyanoethoxy)methyl-2'-deoxyguanine), above, may also recognize 3'-0-2-(cyanoethoxy)methyl -2'-deoxyadenine; 3'-0-2- (cyanoethoxy)methyl -2'-deoxythymine; and 3'-0-2-(cyanoethoxy)methyl -2'-deoxycytosine.
- affinity reagents that differentially recognize RT blocking groups is within the skill of one of ordinary skill in the art guided by this disclosure.
- an antibody is raised against the hapten O- azidomethyl (e.g., -O- azidomethyl or azidomethyl conjugated to keyhole limpet hemocyanin) and positively and negatively screened for binding to a 3'-0-azidomethyl -2'-d M nucleotide at the 3' end of a GDS TP (where N is each of A, T, G or C) and for not binding to 3'-0-X-2'-dNM where O-X is a different blocking group present in the sequencing reaction.
- O- azidomethyl e.g., -O- azidomethyl or azidomethyl conjugated to keyhole limpet hemocyanin
- the hapten may be deoxyribose with the 3'-0 blocking group, a nucleotide (e.g., monophosphate or triphosphate with a 3'-0 blocking group), or the like, so long as the selection process identified affinity reagents with the desired specificity.
- a nucleotide e.g., monophosphate or triphosphate with a 3'-0 blocking group
- sequencing is carried out using four NLRT each having a 3'-0-blocking group in which the blocking groups of 2 or more, alternatively 3 or more, alternatively all 4 are structurally similar in the sense that (1) they have the same number of atoms or the number of atoms differs by no more than a small number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); (2) the molecular formulas of the blocking group moieties differ by 1 to 10 atoms (e.g., single H replaced by CH3 is 3 differences; H replaced by F, O replaced by S), e.g., 1 atom, 2 atoms, 3 atoms, 4 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms or 10 atoms.
- the blocking group moiety may have any of the properties described hereinabove in the section captioned "Properties of Reversible Terminator Blocking Groups and Nucleotides Containing The
- the affinity reagent binds to a NLRT (e.g., 3'-0- azidomethyl-2'-deoxyguanine) but does not bind to the corresponding unblocked nucleotide (e.g., 3'-OH-2'-deoxyguanine).
- a NLRT e.g., 3'-0- azidomethyl-2'-deoxyguanine
- the corresponding unblocked nucleotide e.g., 3'-OH-2'-deoxyguanine
- the affinity reagent binds to a NLRT (e.g., 3'-0-azidomethyl-
- An affinity reagent that specifically recognizes NLRT-A is referred to as antiA.
- An affinity reagent that specifically recognizes NLRT-T is referred to as antiT.
- An affinity reagent that specifically recognizes NLRT-G is referred to as antiG.
- An affinity reagent that specifically recognizes NLRT-C is referred to as antiC.
- An affinity reagent that specifically recognizes NLRT-U is referred to as antiU.
- affinity reagents may be directly labeled.
- affinity reagents may be an unlabeled primary affinity reagent detectable using a labeled secondary affinity reagent.
- an unlabeled primary affinity reagent that specifically binds a NLRT may be detected with a labeled secondary affinity reagent that binds the primary affinity reagent (for example, a labeled antibody that binds the primary affinity reagent). See Section 4.5, below
- the affinity reagent is an antibody. Any method for antibody production that is known in the art may be employed.
- antibody means an immunoglobulin molecule or composition
- antibody e.g., monoclonal and polyclonal antibodies
- genetically engineered forms such as chimeric, humanized and human antibodies, heteroconjugate antibodies (e.g., bispecific antibodies), and antibody fragments.
- the antibody may be from recombinant sources and/or produced in animals, including without limitation transgenic animals.
- the term "antibody” as used herein includes "antibody fragments,” including without limitation Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, nanobodies diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques.
- F(ab')2 fragments can be generated by treating an antibody with pepsin.
- the resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab 1 fragments.
- Papain digestion can lead to the formation of Fab fragments.
- Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
- the antibodies can be in any useful isotype, including IgM and IgG, such as IgGl, lgG2, lgG3 and lgG4.
- the affinity reagents are minibodies.
- Minibodies are engineered antibody constructs comprised of the variable heavy (VH) and variable light (VL) chain domains of a native antibody fused to the hinge region and to the CH3 domain of the immunoglobulin molecule.
- Minibodies are thus small versions of whole antibodies encoded in a single protein chain which retain the antigen binding region, the CH3 domain to permit assembly into a bivalent molecule and the antibody hinge to accommodate dimerization by disulfide linkages.
- a single domain antibody (sdAb) may also be used.
- a single domain antibody, or NANOBODY (Ablynx) is an approximately antibody fragment with a single monomeric variable antibody domain. Single domain antibodies bind selectively to specific antigens and are smaller (MW 12-15 kDa) than conventional antibodies.
- Methods for raising polyclonal antibodies are known and may be used to produce NLRT-specific antibodies. For one approach see Example 2 below.
- a rabbit is injected with NLRT-A (conjugated to an immunogen) to raise antibodies, and antibodies are selected to do not bind to: the same structure lacking the blocking group (e.g., having a 3'-OH), and the other NLRTs (NLRT-T, NLRT-G, and NLRT-C).
- the polyclonal antibodies produced recognize the specific NLRT that is incorporated at the 3' end of a growing DNA chain at a particular position on a sequencing array, but not that same nucleoside at other interior positions of the growing chain or to other NLRTs that may be incorporated elsewhere on the array.
- the polyclonal antibodies may also recognize unincorporated NLRT-A, but unincorporated NLRTs are washed away before incorporated NLRTs are probed using labeled affinity reagents.
- the hapten may be deoxyribose with a 3'-0-blocking group (i.e., no nucleobase) or the 3'-0-blocking group alone.
- antibodies are raised against a polynucleotide with a NLRT of interest at the 3' end.
- antibodies are raised against a polynucleotide annealed to a template molecule.
- antibody producing cells can be harvested from an animal immunized with an immunogen comprising an NLRT and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
- Such techniques are well known in the art (e.g., the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein Nature 256:495- 497, 1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983, Immunol.
- Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a particular RT and the monoclonal antibodies can be isolated.
- Specific antibodies, or antibody fragments, reactive against particular antigens or molecules may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with cell surface components.
- immunoglobulin genes or portions thereof, expressed in bacteria with cell surface components.
- complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (see for example Ward et al., Nature 341:544-546, 1989; Huse et al. Science 246:1275, 1989; and McCafferty et al. Nature 348:552-554, 1990).
- antibodies specific for a target NLRT are readily isolated by screening antibody phage display libraries.
- an antibody phage library is optionally screened by using to identify antibody fragments specific for a target NLRT. Methods for screening antibody phage libraries are well known in the art.
- Anti-NLRT antibodies also may be produced in a cell-free system.
- Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44, 2009; Spirin, Trends Biotechnol. 22: 538-45, 2004; and Endo et al., Biotechnol. Adv. 21: 695-713, 2003.
- Anti-NLRT antibodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides. See Section 8.4, below. Many methods of purifying polypeptides are known in the art. Affinity purification of anti-NLRT antibodies from a polyclonal antiserum are described in Example 2 below.
- Antibodies can be labeled using any methods known in the art. Methods for linking of antibodies and other affinity reagents to reporter molecules, e.g., signal-generating proteins including enzymes and fluorescent/luminescent proteins are well known in the art (Wild, The Immunoassay Handbook, 4 th ed.; Elsevier: Amsterdam, the Netherlands, 2013; Kobayashi and Ova ma, Analyst 136:642-651, 2011).
- An aptamer is an oligonucleotide or peptide molecule that binds to a specific target molecule.
- Aptamers can be classified as: (a) DNA or RNA or XNA aptamers, which consist of (usually short) strands of oligonucleotides; and (b) peptide aptamers, which consist of one (or more) short variable peptide domains, attached at both ends to a protein scaffold.
- Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets, e.g., NLRTs.
- SELEX systematic evolution of ligands by exponential enrichment
- aptamers with affinity for a target NLRT can be selected from a large oligonucleotide library through SELEX, an iterative process in which non-binding aptamers are discarded and aptamers binding to the proposed target are expanded. Initial positive selection rounds are sometimes followed by negative selection. This improves the selectivity of the resulting aptamer candidates. In this process, the target NLRT is immobilized to an affinity column.
- the aptamer library is applied and allowed to bind. Weak binders are washed away and bound aptamers are eluted and amplified using PCR. Then the pool of amplified aptamers is reapplied to the targets. The process is repeated multiple times under increasing stringency until aptamers of the desired selectivity and affinity are obtained. See, e.g., Jayasena, et al., Clinical Chemistry 45:1628-1650, 1999. Peptide aptamer selection can be made using different systems, including the yeast two-hybrid system.
- Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. See, e.g., Reverdatto et al., 2015, Curr. Top. Med. Chem. 15:1082-1101.
- Affimers are small (12-14 kDa), highly stable proteins that bind their target molecules with specificity and affinity similar to that of antibodies. These proteins share the common tertiary structure of an alpha-helix lying on top of an anti-parallel beta-sheet. Affimer proteins display two peptide loops and an N-terminal sequence that can all be randomised to bind to desired target proteins with high affinity and specificity in a similar manner to monoclonal antibodies. Stabilisation of the two peptides by the protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides.
- Affimers specific for a NLRT can be selected by the use of phage display libraries that are screened to identify an Affimer protein with high-specificity binding to the target NLRT and high binding affinities (e.g., in the nM range).
- Many different labels, tags and fusion proteins, such as fluorophores, have been conjugated to Affimer proteins for use in various applications. See, e.g., US Pat. No. 8,481,491, US 8,063,019, and WO 2009/136182, which are incorporated herein by reference. See also Crawford et al., Brief Fund. Genomic Proteomic, 2:72-79, 2003.
- knottin or “inhibitor cystine knot” (ICK) is a protein structural motif containing three disulfide bridges. Along with the sections of polypeptide between them, two disulfides form a loop through which the third disulfide bond (linking the third and sixth cysteine in the sequence) passes, forming a knot. New binding epitopes can be introduced into natural knottins using protein engineering, and knottins have been engineered to target a broad range of targets.
- One approach to production of knottins that are specific for NLRTs is to create and screen knottin libraries using yeast surface display and fluorescence-activated cell sorting.
- knottins with selectivity and high affinity for a target NLRT and labeling such knottins for use in conection with the present invention
- Kintzing and Cochran Curr. Opin. Chem. Biol. 34:143-150, 2016; Moore et al., Drug Discovery Today: Technologies 9(l):e3-ell, 2012; and Moore and Cochran, Meth. Enzymol. 503:223-51, 2012.
- Labeled affinity reagents can be used to sequence a template nucleic acid by a variety of methods. They can also be used in a variety of applications other than sequencing, as will be apparent to those of skill in the art. Any method of labeling antibodies and other affinity reagents of the invention may be used. 4.3.1 Fluorescent Detectable Labels
- affinity reagents used in the practice of the invention can be detectably labeled.
- affinity reagents described herein can be detectably labeled with fluorescent dyes or fluorophores.
- fluorescent dye means to a fluorophore (a chemical compound that absorbs light energy of a specific wavelength and re-emits light at a longer wavelength).
- Fluorescent dyes typically have a maximal molar extinction coefficient at a wavelength between about 300 nm to about 1,000 nm or of at least about 5,000, more preferably at least about 10,000, and most preferably at least about 50,000 cm-1 M-l, and a quantum yield of at least about 0.05, preferably at least about 0.1, more preferably at least about 0.5, and most preferably from about 0.1 to about 1.
- the literature also includes references providing lists of fluorescent molecules, and their relevant optical properties for choosing fluorophores or reporter-quencher pairs, e.g., Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 2005); and the like. Further, there is extensive guidance in the literature for derivatizing reporter molecules for covalent attachment via common reactive groups that can be added to an RT or portion thereof, as exemplified by: Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al., U.S. Pat. No. 4,351,760; and the like. Each of the aforementioned publications is incorporated herein by reference in its entirety for all purposes.
- Exemplary fluorescent dyes include, without limitation, acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, and rhodamine dyes.
- Exemplary fluorescent dyes include, without limitation, fluorescein, FITC, Texas Red, ROX, Cy3, an Alexa Fluor dye (e.g., Alexa Fluor 647 or 488), an ATTO dye (e.g., ATTO 532 or 655), and Cy5.
- Exemplary fluorescent dyes can further include dyes that are used in, or compatible with, two- or four-channel SBS chemistries and workflows.
- Exemplary label molecules may be selected from xanthene dyes, including fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are widely available commercially with substituents on their phenyl moieties which can be used as the site for linking to an affinity reagent.
- Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are l-dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate, and 2-p-toluidinyl-6-naphthalene sulfonate.
- Other labels include 3-phenyl-7- isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2- benzoxazolyl)phenyl)maleimide; benzoxadiazoles; stilbenes; pyrenes; and the like.
- labels are selected from fluorescein and rhodamine dyes.
- Fluorophores that can be used as detectable labels for affinity reagents or nucleoside analogues include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, VicTM, LizTM, TamraTM, 5-FamTM, 6-FamTM, 6-HEX, CAL Fluor Green 520, CAL Fluor Gold 540, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 615, CAL Fluor Red 635, and Texas Red (Molecular Probes).
- the affinity reagent e.g., antibody or affimer
- the enzyme associated with an affinity reagent bound to a primer extension product produces a detectable signal.
- enzymes include peroxidase, phosphatase, luciferase, etc.
- the enzyme is a peroxidase.
- the affinity reagent (e.g., antibody or affimer) is directly labeled enzymatically
- an antibody or other affinity reagent is labeled using peroxidase, such as horseradish peroxidase (HRP) or a phosphatase, such as an alkaline phosphatase (Beyzavi et al., Annals Clin Biochem 24:145-152, 1987).
- HRP horseradish peroxidase
- a phosphatase such as an alkaline phosphatase
- the affinity reagent is coupled to (or is part of a fusion protein with) luciferase or other protein that can be used to produce a chemiluminescent signal.
- the affinity reagent can be coupled/fused to an enzyme system that is selected to produce a non- optical signal, such as a change in pH where protons can be detected, for example, by ion semiconductor sequencing (e.g., Ion Torrent sequencers; Life Technologies Corporation, Grand Island, NY).
- ion semiconductor sequencing e.g., Ion Torrent sequencers; Life Technologies Corporation, Grand Island, NY.
- Use of enzyme labeled affinity reagents has certain advantages, including high sensitivity resulting from signal amplification and the ability to tailor the sequencing method to a variety of instruments. Enzyme reporter systems are reviewed in Rashidian et al., Bioconjugate Chem. 24:1277-1294, 2013.
- fusions directly linking recombinant antibody fragments e.g., single- chain Fv fragments (scFvs) with reporter proteins
- scFvs single- chain Fv fragments
- reporter proteins Skerra and Pliickthun, Science 240:1038- 1041, 1988; Bird et al., Science 242:423-426, 1988; Huston et al., Methods Enzymol 203:46-88, 1991; Ahmad et al., Clin. Dev. Immunol. 2012:1, 2012
- photoproteins with bioluminescent properties may be used as reporter proteins in fusion proteins with antibody fragments, epitope peptides and streptavidin, for example (Oyama et al., Anal Chem 87:12387-12395, 2015; Wang et al., Anal Chim Acta 435:255-263, 2001; Desai et al., Anal Biochem 294:132-140, 2001; Inouye et al., Biosci Biotechnol Biochem 75:568-571, 2011).
- bioluminescent properties e.g., luciferases and aequorin
- An affinity reagent may be directly labeled (e.g., by conjugation to the label, e.g., via a covalent bond, to a fluorophore) or indirectly labeled, e.g., by binding of a labeled secondary affinity reagent that binds a primary affinity reagent directly bound to the extended primer with a 3' NLRT.
- Unlabeled primary affinity reagents bind the target nucleotide and labeled secondary affinity reagents (e.g., antibodies, aptamers, affimers or knottins) bind the primary affinity reagents.
- the primary and/or secondary affinity reagent is an antibody.
- the affinity reagent is a "primary" antibody (e.g., rabbit anti-NLRT-C antibody) and the secondary binder is a labeled anti-primary antibody (e.g., dye-labeled goat anti-rabbit antibody).
- a secondary affinity reagent provides advantageous signal amplification.
- the assay comprises two distinct parts: first, there is a period of incubation (usually one hour) with the unlabeled primary antibody, during the antibody binds to the antigen (assuming of course that the antigen is present). Excess unbound primary antibody is then washed away and a labeled secondary reagent is added. After a period of incubation (again one hour), excess secondary reagent is washed away and the amount of label associated with the primary antibody (i.e., indirectly via the secondary reagent) is quantified.
- the label usually results in the production of a colored substance or an increase in the amount of light emitted at a certain wavelength, if the antigen is present.
- Primary and secondary antibodies may be selected to distinguish multiple antigens (e.g., to distinguish RT-A, RT-C, RT-G and RT-T from each other), unlabeled primary antibodies (typically monoclonal or engineered antibodies) may have different isotypes and/or have sequences characteristic of different species (e.g., polyclonal antibodies raised in different animals or corresponding monoclonal antibodies or other affinity reagents).
- labeled secondary (i.e., anti-primary) antibodies for each antigen be specific for the appropriate isotype or species sequence.
- primary antibodies of isotypes IgGl, lgG2a, lgG2b, and lgG3 can be used with isotype-specific secondary antibodies.
- Primary and secondary antibodies or other agents may be added to a sequencing array sequentially, simultaneously, may be precombined under conditions in which the secondary antibody(s) bind to the primary antibody and added to the array as a complex. See Figure 2 and Example 7.
- Sequencing using methods of the invention may be two-, three-, or four-color sequencing.
- each affinity reagent is directly or indirectly labeled with a different detectable label (e.g., a fluorescent dye) or combination of labels producing a unique signal.
- a detectable label e.g., a fluorescent dye
- a portion (e.g., 50%) of the affinity reagent molecules specific for the single antigen can be labeled with one dye and another portion (e.g., 50%) of the affinity reagent molecules specific for the single antigen can be labeled with the other dye.
- an array that comprises single- stranded nucleic acid templates disposed at positions on a surface. Sequencing by extension, or SBS, is performed in order to determine the identity of nucleotides at detection positions in nucleic acid templates in multiple sequencing cycles by: (i) binding (or incorporating) an unlabeled complementary nucleotide (NLRT) to a nucleotide at a detection position, (ii) labeling the NLRT by binding to it a directly or indirectly labeled affinity reagent that specifically binds to such an NLRT; (iii) detecting the presence or absence of a signal(s) associated with the complementary NLRT at the detection position, the signal resulting from the label (e.g., a fluorescent signal); wherein (1) detecting a first signal and not a second signal at the detection position identifies the complementary NLRT as selected from NLRT-A, NLRT-T, NLRT-G and NLRT-C; (2)
- Another such method comprises: providing a plurality of nucleic acid templates each comprising a primer binding site and, adjacent to the primer binding site, a target nucleic acid sequence; performing sequencing reactions on the plurality of different nucleic acid templates by hybridizing an primer to the primer binding site and extending individual primers by one nucleotide per cycle in one or more cycles of sequencing-by-synthesis using a set of NLRTs and a corresponding set of affinity reagents, e.g.: (i) first NLRTs and first affinity reagents that specifically bind to the first NLRTs and that comprise a first label; (ii) second NLRTs and second affinity reagents that specifically bind to the second NLRTs and that comprise a second label; (iii) third NLRTs and third affinity reagents that specifically bind to the third NLRTs and that comprise both the first label and the second label; and (iv) fourth NLRTs and fourth affinity reagents that specifically bind to
- the affinity reagents include a detectable label that is present at distinguishable intensities.
- a method comprises: providing a plurality of nucleic acid templates each comprising a primer binding site and, adjacent to the primer binding site, a target nucleic acid sequence; performing sequencing reactions on the plurality of different nucleic acid templates by hybridizing a primer to the primer binding site and extending individual primers by one nucleotide per cycle in one or more cycles of sequencing- by-synthesis using a set of NLRTs and a corresponding set of affinity reagents, e.g.: (i) first NLRTs and first affinity reagents that specifically bind to the first NLRTs and that comprise a label at a first intensity; (ii) second NLRTs and second affinity reagents that specifically bind to the second NLRTs and that comprise the label at a second intensity; (iii) third NLRTs
- affinity affinity reagents are used that are labeled with one or the same number of molecules of a single dye yet discriminate among the four NLRTs as a result of different binding efficiencies (i.e., the average number of affinity reagents that are bound to a single spot on an array, e.g., 10% of all copies of the target DNA molecule for NLRT- A, 30% for NLRT-T, and 60% for NLRT-C (and zero percent or little detectable binding for NLRT- G).
- the targets have the same blocking group and affinity reagents are selected that have different affinities for their target.
- blocking groups may be modified with small chemical changes to tune the efficiency of binding of the same affinity reagent, thus generating base specific levels of signal.
- an unmodified blocking group may produce the highest signal (100% of signal)
- a blocking group with modification 1 may produce a lower level of signal (e.g. 50%)
- a blocking group with modification 2 may produce a still lower signal with even less (25%), etc.
- Affinity Agent 1, color 1, low intensity has an signal intensity close to zero and Affinity Agent 2, color 2, low intensity has a higher signal intensity (25-40%).
- 2-color sequencing can be carried out in which a single species of nucleotide is uses as a mixture of nucleotides in which a portion are labeled with one blocking group and the remainder are labeled with the other blocking group.
- a single species of nucleotide is uses as a mixture of nucleotides in which a portion are labeled with one blocking group and the remainder are labeled with the other blocking group.
- nucleotides having blocking group 2 cyanoethoxymethyl-prime - dT Mixture with 30% of nucleotides having blocking group
- the antibody could recognize two bases (a nucleotide dimer) where the downstream base is modified with the addition of a cleavable or un-cleavable group.
- the last-incorporated base is identified by the binding of two affinity reagents in combination: one affinity reagent specifically recognizes and binds to the nucleobase, and the second affinity reagent specifically recognizes and binds to the blocking group. Only when both affinity reagents bind and/or are in spatial proximity, can a determination of the identity of the terminal base be made such as when the two affinity reagents include a FRET donor-acceptor pair as their respective "labels.” Alternatively, the binding of one of the affinity reagents could lead to a conformational change that allows or enhances binding of the second affinity reagent.
- nucleoside analogues described herein can be used in a variety of sequencing methods.
- the analogues can be used in one label (sometimes called "no-label"), two-label, three-label, or four-label sequencing methods, in which unlabeled analogues are paired with affinity reagents directly or indirectly labeled according to a one-, two-, three-, or four-label scheme.
- Exemplary one-label sequencing methods include, but are not limited to, methods in which nucleoside analogues having different nucleobases [e.g., A, C, G, T) are delivered in succession and incorporation is detected by detecting the presence or absence of the same signal or label for each different nucleobase.
- nucleoside analogues having different nucleobases e.g., A, C, G, T
- incorporation is detected by detecting the presence or absence of the same signal or label for each different nucleobase.
- one-label methods are sometimes known as one-color methods because the detection signal and/or label is the same for all nucleobases, even though it may differ in intensity (or be absent) for each nucleoside analogue.
- incorporation of a nucleoside into a primer by DNA polymerase mediated template directed polymerization can be detected by detecting a pyrophosphate cleaved from the nucleoside pyrophosphate.
- Pyrophosphate can be detected using a coupled assay in which ATP sulfurylase converts pyrophosphate to ATP, in the presence of adenosine 5' phosphosulfate, which in turn acts as a substrate for luciferase-mediated conversion of luciferin to oxyluciferin, generating visible light in amounts proportional to ATP generation.
- two-label, or two-color, sequencing can be performed using the RTs and affinity reagents described herein, using two distinguishable signals in a combinatorial fashion to detect incorporation of four different RTs.
- Exemplary two- label systems, methods, and compositions include, without limitation, those described in U.S. Pat. No. 8,617,811, the contents of which are hereby incorporated by reference in the entirety for all purposes and particularly for disclosure related to two-label sequencing.
- incorporation of a first RT e.g., RT-A
- a first affinity reagent that includes a first label
- Incorporation of a second RT is detected by labeling the second RT by specific binding of a second affinity reagent that includes a second label, then detecting the presence of the second label.
- Incorporation of a third RT is detected by labeling the third RT by specific binding of a third affinity reagent that includes both the first and the second label, then detecting the presence of both the first and second label; and, incorporation of a fourth RT [e.g., RT-G) is detected by detecting the absence of both first and second labels, whether this results from binding of a fourth affinity reagent that is unlabeled or from the fact that no fourth affinity reagent is included in the affinity reagent set that is used.
- the first label is distinguishable from the second label and the combination of the first and second label can be distinguished from the first and second label taken alone.
- three-label sequencing can be performed using a first RT labeled by specific binding of an first affinity reagent that includes a first label, a second RT labeled by specific binding of an second affinity reagent that includes a second label, a third RT labeled by specific binding of a third affinity reagent that includes a third label.
- the corresponding affinity reagent is omitted from the affinity reagent set, or is unlabeled, or includes a combination of two or more of the first, second, and third labels (or a mixture of affinity reagents that are labeled with a different one of the labels and that specifically bind to the fourth RT).
- the first, second and third labels are distinguishable from each other.
- four-label sequencing can employ a first NLRT that is labeled by specific binding of a first affinity reagent that includes a first label, a second NLRT that is labeled by specific binding of a second affinity reagent that includes a second label, a third NLRT that is labeled by specific binding of a third affinity reagent that includes a third label, and a fourth NLRT that is labeled by specific binding of a fourth affinity reagent that includes a fourth label.
- the first, second, third and fourth labels are distinguishable from each other.
- Affinity reagents that recognize different epitopes of a single NLRT may be used in combination.
- a first affinity reagent that recognizes the nucleobase portion of the incorporated NLRT may be used with a second affinity reagent that recognizes a blocking group. Staining may be done simultaneously or sequentially. In sequential staining the second affinity reagent may be applied while the first affinity reagent remains bound to the NLRT or after removal of the first affinity reagent in the case of re-probing (discussed below).
- affinity reagent sets are used to label RTs used in SBS. For example, in one embodiment, for an RT set that includes four RTs (RT-A, RT-T, RT-C and RT-G), there could be a corresponding affinity reagent set of four affinity reagents, each specifically recognizing and binding to one of the RTs (antiA, antiT, antiC and antiG). Affinity reagent sets describe combinations of affinity reagents that can be (i) provided in kit form, as a mixture or in separate containers and/or (ii) contacted with, or combined on, a sequencing array (e.g., within a sequencing flow cell). [0186] According to one embodiment, each member of an affinity reagent set has a different, distinguishable detectable label, as in four-color SBS.
- one member of an affinity reagent set is unlabeled, while the other members are labeled.
- the affinity reagent set could simply exclude the unlabeled affinity reagent and include only the labeled affinity reagents.
- one affinity reagent is labeled with a first label (e.g., antiA); a second affinity reagent is labeled with a second label (e.g., antiT); a third affinity reagent is labeled with a third label (e.g., antiC); and a fourth affinity reagent is unlabeled or simply excluded from the affinity reagent set (e.g., antiG).
- a first label e.g., antiA
- a second affinity reagent is labeled with a second label
- a third affinity reagent is labeled with a third label (e.g., antiC)
- a fourth affinity reagent is unlabeled or simply excluded from the affinity reagent set (e.g., antiG).
- affinity reagent set would be useful for three-color sequencing.
- one affinity reagent (e.g., antiA) is labeled with a first label; a second affinity reagent (e.g., antiT) is labeled with a second label; a third affinity reagent (e.g., antiC) is labeled with both the first label and the second label; and a fourth affinity reagent (e.g., antiG) is unlabeled (or excluded from the affinity reagent set).
- the third affinity reagent may include a mixture of affinity reagent molecules, all of which specifically bind to a particular base (e.g., all are antiC), but some include the first label and some include the second label.
- affinity reagent sets would be useful for two-color sequencing.
- only a single detectable label is used (or a single combination of two or more labels), but differs in intensity among members of the set, such as when the affinity reagent includes differing amounts of the label (or of at least one label of a combination of two or more labels).
- a first affinity reagent e.g., antiA
- a second affinity reagent e.g., antiT
- a third affinity reagent e.g., antiC
- a fourth affinity reagent e.g., antiG is unlabeled (or the fourth affinity reagent is excluded from the affinity reagent set).
- a first affinity reagent (e.g., antiA) is labeled with a first label at a first intensity and a second label; a second affinity reagent (e.g., antiT) is labeled with the same first label but at a second intensity and the same second label; a third affinity reagent (e.g., antiC) is labeled with the same first label but at a third intensity and the same second label; and a fourth affinity reagent (e.g., antiG) is unlabeled, is labeled only with the second label, or is excluded from the affinity reagent set.
- Nucleoside analogues e.g., NLRTs
- oligo- or polynucleotides containing such nucleoside analogues or reaction products thereof can be used as a component of a reaction mixture.
- such components can be used in reaction mixtures for nucleic acid sequencing [e.g., SBS).
- Exemplary reaction mixtures include, but are not limited to, those containing (a) template nucleic acid; (b) polymerase; (c) oligonucleotide primer; (d) a 3'-0 reversibly blocked nucleoside analogue, or a mixture of 3'-0 reversibly blocked nucleoside analogues having structurally different nucleobases; and (e) a labeled affinity reagent.
- Exemplary sequencing reaction mixtures of the invention include, but are not limited to, arrays comprising a plurality of different template nucleic acids immobilized at different locations on the array; (b) polymerase; (c) oligonucleotide primer; (d) and one or a mixture of NLRTs.
- Exemplary sequencing reaction mixtures of the invention include, but are not limited to, arrays comprising a plurality of different template nucleic acids immobilized at different locations on the array; (b) growing DNA strands (GDS) (which may comprise a 3' NLRT; and (c) one or more affinity reagents (e.g., an affinity reagent set as described hereinabove).
- GDS DNA strands
- affinity reagents e.g., an affinity reagent set as described hereinabove.
- the template polynucleotide is DNA [e.g., cDNA, genomic DNA, transcriptome or microbiome DNA, amplification products, etc.) or RNA.
- the polynucleotide is either double stranded or single stranded.
- the template nucleic acid is immobilized on a solid surface.
- the template nucleic acid is immobilized on a substrate [e.g., a bead, flow cell, pad, channel in a microfluidic device and the like).
- the substrate may comprise silicon, glass, gold, a polymer, PDMS, and the like.
- the template nucleic acid is immobilized or contained within a droplet (optionally immobilized on a bead or other substrate within the droplet).
- the template nucleic acid is an immobilized DNA concatemer comprising multiple copies of a target sequence.
- the template nucleic acid is represented as a DNA concatemer, such as a DNA nanoball (DNB) comprising multiple copies of a target sequence and an "adaptor sequence". See PCT Pat. Pub. WO 2007/133831, the content of which is hereby incorporated by reference in its entirety for all purposes.
- the template is a single polynucleotide molecule.
- the template is present as a clonal population of template molecules (e.g., a clonal population produced by bridge amplification or Wildfire amplification).
- the method is not limited to a particular form of template, and the template can be any template such as, for example, a DNA concatemer, a dendrimer, a clonal population of templates [e.g., as produced by bridge amplification or Wildfire amplification) or a single polynucleotide molecule.
- a template can alternatively refer to a concatemer template, a dendrimer, a clonal population of, e.g., short linear templates, a single molecule template (e.g., in a zero-mode waveguide), and templates in other forms.
- Suitable template nucleic acids including DNBs, clusters, polonys, and arrays or groups thereof, are further described in U.S. Pat. Nos. 8,440,397; 8,445,194; 8,133,719; 8,445,196; 8,445,197; 7,709,197; 12/335,168, 7,901,891; 7,960,104; 7,910,354; 7,910,302; 8,105,771; 7,910,304; 7,906,285; 8,278,039; 7,901,890; 7,897,344; 8,298,768; 8,415,099; 8,671,811; 7,115,400; 8,236,499, and U.S. Pat. Pub. Nos. 2015/0353926; 2010/0311602; 2014/0228223; and 2013/0338008, all of which are hereby incorporated by reference in their entirety.
- the invention provides a DNA array comprising: a plurality of template DNA molecules, each DNA molecule attached at a position of the array, a complementary DNA sequence base-paired with a portion of the template DNA molecule at a plurality of the positions, wherein the complementary DNA sequence comprises at its 3' end an incorporated first reversible terminator deoxyribonucleotide; and a first affinity reagent bound specifically to at least some of the first reversible terminator deoxyribonucleotides.
- the DNA array comprises primer extension products with 3' terminal nucleotides comprising A, T, G or C nucleobases or analogs thereof, and affinity reagents bound to the primer extension products.
- Kite may be provided for practicing the invention. As described above, NLRTs and
- kits comprising both NLRTs and NLRT sets and affinity reagents or affinity reagent sets.
- the invention provides kite that include, without limitation (a) a reversible terminator nucleotide (RT) or RT set that includes one, two, three, four or more different individual RTs; (b) a corresponding affinity reagent or affinity reagent set that includes one, two, three, four or more affinity reagents, each of which is specific for one of the RTs; and (c) packaging materials and or instructions for use.
- RT reversible terminator nucleotide
- a corresponding affinity reagent or affinity reagent set that includes one, two, three, four or more affinity reagents, each of which is specific for one of the RTs
- packaging materials and or instructions for use are packaging materials and or instructions for use.
- such a kit comprises a plurality of the RTs, wherein each RT comprises a different nucleobase, and a plurality of affinity reagents, wherein each affinity reagent binds specifically to one of the RTs.
- the invention provide a kit comprising (a) a reversible terminator nucleotide as herein described that may be incorporated into a primer extension product; (b) a first affinity reagent that is binds specifically to the reversible terminator nucleotide when incorporated at the 3' terminus of a primer extension product; and (c) packaging for (a) and (b).
- the kit contains a plurality of reversible terminator deoxyribonucleotides, wherein each reversible terminator deoxyribonucleotide comprises a different nucleobase, and a plurality of first affinity reagents, wherein each first affinity reagent binds specifically a different one of the reversible terminator deoxyribonucleotides.
- the first affinity reagents are detectably labeled and can be distinguished from each other.
- the kit comprises secondary affinity reagents.
- the first and/or second affinity reagents are antibodies.
- the reversible terminator deoxyribonucleotide has the structure of Formula I:
- R 1 is a 3'-0 reversible blocking group
- R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof
- R 3 comprises of one or more phosphates.
- NLRTs, kits and methods described herein may be used in many other applications, such as detecting what is at the end of naturally or experimentally fragmented DNA (or in DNA gaps); capturing oligonucleotides or polynucleotides with a specific end-base (end of molecule or end within the gap of a strand) with or without specific modification. Both 5' or 3' end/gap bases may be detected. Affinity reagents may be used for ligation, hybridization, and other detection.
- Removal of blocking groups and affinity reagents can occur simultaneously.
- an array is exposed to conditions in which of blocking groups and affinity reagents are removed simultaneously.
- the array is contacted with a solution with a combination of agents some of which result in removal of the affinity reagents (e.g., high salt, small molecule competitors, protease, etc.) combined with agents that cleave the blocking group.
- agents some of which result in removal of the affinity reagents e.g., high salt, small molecule competitors, protease, etc.
- removal of the 3' blocking group results in removal of the affinity reagent. Without intending to be bound by a particular mechanism, it is believed that in these cases, removal of the blocking moiety destroys the epitope required for binding of the antibody or other affinity reagent.
- Nucleoside analogues or NLRTs include those that are 3'-0 reversibly blocked.
- the blocking group provides for controlled incorporation of a single 3'-0 reversibly blocked NLRT at the 3'-end of a primer, e.g., a GDS extended in a previous sequencing cycle.
- a azidomethyl blocking group can be removed by treatment with phosphine (a widely used process) and an antibody affinity reagent can be removed by treatment with a low pH (e.g., 100 mM glycine pH 2.8) or high pH (e.g., 100 mM glycine pH 10), high salt, or chaotropic stripping buffer.
- a single treatment or condition can be used to remove both the NLRT and the affinity reagent (e.g., phosphine in a high salt buffer).
- removal of the blocking group results in disassociation of the affinity reagent if, for example, the blocking group is required for affinity reagent binding.
- the 3'-0 reversible blocking group can be removed by enzymatic cleavage or chemical cleavage (e.g., hydrolysis).
- the conditions for removal can be selected by one of ordinary skill in the art based on the descriptions provided herein, the chemical identity of the blocking group to be cleaved, and nucleic acid chemistry principles known in the art.
- the blocking group is removed by contacting the reversibly blocked nucleoside with a reducing agent such as dithiothreitol (DTT), or a phosphine reagent such as tris(2- carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THP), or tris(hydroxypropyl) phosphine.
- a reducing agent such as dithiothreitol (DTT)
- a phosphine reagent such as tris(2- carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THP), or tris(hydroxypropyl) phosphine.
- the blocking group is removed by washing the blocking group from the incorporated nucleotide analogue using a reducing agent such as a phosphine reagent.
- the blocking group is photolabile, and the blocking group can be removed by application of, e.g., UV light
- the blocking group can be removed by contacting the nucleoside analogue with a transition metal catalyzed reaction using, e.g., an aqueous palladium (Pd) solution. In some cases, the blocking group can be removed by contacting the nucleoside analogue with an aqueous nitrite solution. Additionally, or alternatively, the blocking group can be removed by changing the pH of the solution or mixture containing the incorporated nucleotide analogue. For example, in some cases, the blocking group can be removed by contacting the nucleoside analogue with acid or a low pH [e.g., less than 4) buffered aqueous solution. As another example, in some cases, the blocking group can be removed by contacting the nucleoside analogue with base or a high pH [e.g., greater than 10) buffered aqueous solution.
- a transition metal catalyzed reaction using, e.g., an aqueous palladium (Pd) solution. In
- 3'-0 reversible blocking groups that can be cleaved by a reducing agent, such as a phosphine include, but are not limited to, azidomethyl.
- 3'-0 reversible blocking groups that can be cleaved by UV light include, but are not limited to, nitrobenzyl.
- 3'-0 reversible blocking groups that can be cleaved by contacting with an aqueous Pd solution include, but are not limited to, allyl.
- 3'-0 reversible blocking groups that can be cleaved with acid include, but are not limited to, methoxymethyl.
- 3'-0 reversible blocking groups that can be cleaved by contacting with an aqueous buffered (pH 5.5) solution of sodium nitrite include, but are not limited to, aminoalkoxyl.
- Antibody-based affinity reagents can be removed by low pH, high pH, high or low salt, or denaturing agents such as a chaotropic stripping buffer.
- Other classes of affinity reagents e.g., aptamers
- affinity reagents such as antibodies
- affinity reagents can be removed by introducing an agent that competes with the bound epitope for affinity reagent binding, for example as illustrated in Example 10 below.
- affinity reagents may also be removed by disrupting the ability of the agent to bind the incorporated NLRT. Typically this occurs when the 3' blocking group is cleaved from the incorporated nucleotide analog.
- affinity reagent binding depends on the presence of the blocking group (for example, in cases in which an epitope recognized by a 1° antibody includes the blocking group or a portion thereof) removal of the blocking group results in release of the affinity reagent as well.
- an affinity reagent may be removed without removing the
- Detection e.g., of a fluorescent signal
- detection can be performed either before or after removal of the blocking group as long as the labeled affinity reagent remains bound.
- small compounds are not sufficiently complex by themselves to induce an immune response or be processed in a manner that elicits production of specific antibodies.
- immunogenic carrier proteins such as keyhole limpet hemocyanin (KLH).
- Adjuvants can be mixed and injected with an immunogen to increase the intensity of the immune response.
- Carrier protein conjugation, use of adjuvants and other issues relating to preparation of samples for injection are described in this section on antibody production. Standard procedures for generating, purifying and modifying antibodies for use as antigen-specific probes may be used. See, e.g., Harlow and Lane, "Antibodies: A Laboratory Manual” (1988) and Harlow and Lane, "Using Antibodies: A Laboratory Manual” (1999).
- Haptens Small molecules that are used as antigens are referred to as haptens.
- Haptens are able to act as recognition sites for production of specific antibodies but cannot by themselves stimulate the necessary immune response. Haptens can be made immunogenic by coupling them to a suitable carrier molecule.
- Epitopes An epitope is the specific site on an antigen to which an antibody binds. For very small antigens, practically the entire chemical structure may act as a single epitope. Depending on its complexity and size, an antigen may effect production of antibodies directed at numerous epitopes. Polyclonal antibodies are mixtures of serum immunoglobulins and collectively are likely to bind to multiple epitopes on the antigen.
- KLH Keyhole Limpet Hemocvanin
- KLH Keyhole limpet hemocyanin
- Bovine Serum Albumin Bovine serum albumin (BSA; 67kDa) belongs to the class of serum proteins called albumins.
- Immunization Schedule for Mice Day 0: Collect pre-immune serum from the mouse to use as a blank when performing ELISA screening after immunization. Store frozen. Inject 50 to 100 ug of immunogen (equal to 100 to 200 ⁇ of antigen-adjuvant mixture) per mouse. Typical routes of injection include intraperitoneal (i.p.) or subcutaneous (s.c). One or two such injections may be made per animal. Day 14: Boost with an equivalent amount of immunogen in adjuvant. Day 21: Test bleed and assay antibody response by ELISA.
- mice are bled under anesthesia through the tail vein or the retro-orbital plexis.
- Boost again if necessary.
- Boost again if necessary.
- Immunization Schedule for Rabbits Day 0: Collect pre-immune serum from the rabbit to use as a blank when performing ELISA after immunization. Store frozen. Inject 100 ug of immunogen (equal to about 200 ⁇ of the antigen adjuvant mixture) into each of 8 to 10 subcutaneous sites on the back of the rabbit. Other routes of injection may also be used, but this is by far the easiest with the rabbit. Day 14: Boost with an equivalent amount of adjuvant. Day 21: Test bleed and assay antibody response by ELISA. (Typically, rabbits are bled through the ear vein without anesthetic). It is not difficult to collect 5 to 10 mL of blood, which is more than adequate for measuring antibody response. Day 28: Boost again if necessary. Continue with a similar schedule of alternating boosts and test bleeds until a satisfactory response is observed.
- Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized. These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species.
- a genetically engineered recombinant form of Protein A and G, called Protein A/G is also available. These antibody-binding proteins are available immobilized to beaded agarose resin.
- Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by a variety of methods. Selective precipitation is perhaps the simplest method for separating one type of macromolecule from another.
- Most purification methods involve some form of chromatography whereby molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase).
- Gel filtration also called size- exclusion chromatography or SEC
- SEC size- exclusion chromatography
- ion exchange chromatography molecules are separated according to the strength of their overall ionic interaction with a solid phase material (i.e., nonspecific interactions).
- affinity chromatography also called affinity purification
- a particular ligand is chemically immobilized or “coupled” to a solid support so that when a complex mixture is passed over the column, those molecules having specific binding affinity to the ligand become bound. After other sample components are washed away, the bound molecule is stripped from the support, resulting in its purification from the original sample.
- Antibodies can be covalently modified in many ways to suit the purpose of a particular assay.
- Many immunological methods involve the use of labeled antibodies and a variety of reagents have been created to allow labeling of antibodies. Enzymes, biotin, fluorophores and radioactive isotopes are all commonly used to provide a detection signal in biological assays. Understanding the functional groups available on an antibody is the key to choosing the best method for modification, whether that be for labeling, crosslinking or covalent immobilization.
- Most antibody labeling strategies use one of three targets: (1) Primary amines (-NH2): these occur on lysine residues and the N-terminus of each polypeptide chain.
- -NH2 Primary amines
- Sulfhydryl groups (-SH): these occur on cysteine residues and exist as disulfide bonds that stabilize the whole-molecule structure. Hinge-region disulfides can be selectively reduced to make free sulfhydryls available for targeted labeling.
- Carbohydrates (sugars): glycosylation occurs primarily in the Fc region of antibodies (IgG). Component sugars in these polysaccharide moieties that contain cis-diols can be oxidized to create active aldehydes (-CHO) for coupling.
- Antibody Labeling Methods Any known method for labeling antibodies may be used in the practice of the present invention.
- Antibodies like all proteins are composed of amino acids, and the side chain of lysine, which terminates in a primary amine (-NH2), is commonly used to link labels covalently to antibody molecules.
- -NH2 primary amine
- NHS esters In the case of fluorescent dye labels it is usual to purchase an activated form of the label with an inbuilt NHS ester (also called a 'succinimidyl ester'). The activated dye can be reacted under appropriate conditions with antibodies (all of which have multiple lysine groups). Excess reactive dye is removed by one of several possible methods (often column chromatography) before the labeled antibody can be used in an immunoassay.
- Carbodiimides These reagents (EDC is one very common example) are used to create covalent links between amine- and carboxyl-containing molecules. Carbodiimides activate car boxy I groups, and the activated intermediate is then attacked by an amine (e.g. provided by a lysine residue on an antibody). Carbodimides are commonly used to conjugate antibodies to car boxy lated particles (e.g. latex particles, magnetic beads), and to other car boxy lated surfaces, such as microwell plates or chip surfaces. Carbodiimides are rarely used to attach dyes or protein labels to antibodies, although they are important in the production of NHS-activated dyes (see above).
- EDC is one very common example
- Carbodiimides activate car boxy I groups, and the activated intermediate is then attacked by an amine (e.g. provided by a lysine residue on an antibody).
- Carbodimides are commonly used to conjugate antibodies to car boxy lated particles (e.g. latex particles, magnetic beads), and to other car
- lysines primary amines
- the lone drawback to this labeling strategy is that it occasionally causes a significant decrease in the antigen-binding activity of the antibody. The decrease may be particularly pronounced when working with monoclonal antibodies or when attempting to add a high density of labels per antibody molecule.
- affinity reagent e.g., same primary antibody. More often, a different affinity reagent is used, such as a different antibody preparation (e.g., a different monoclonal antibody), a different class of affinity reagent (e.g., probing with an antibody in the first-halfcycle and with an aptamer in the second-half cycle), or an affinity reagent with a different specificity.
- a different antibody preparation e.g., a different monoclonal antibody
- a different class of affinity reagent e.g., probing with an antibody in the first-halfcycle and with an aptamer in the second-half cycle
- an affinity reagent with a different specificity e.g., in the first-half cycle an array may be probed with anti-A, anti-T, anti-C and anti-G, and in the second-halfcycle the array may be probed with anti-purine and anti-pyrimidine used.
- NLRTs are blocked using two blocking groups, e.g., azidomethyl-T, azidomethyl-G, cyanoethenyl-C and cyanoethenyl-A and the array is probed once with two affinity reagents (one specific for 3'-0-azidomethyl-2'-deoxyribose and the other specific for 3'-0-cyanoethenyl-2'-deoxyribose) and probed a second time with a different pair of affinity reagents (one specific for purines and one specific for pyrimidines).
- An address on an array that shows signal characteristic of 3'-0-azidomethyl-2'-deoxyribose and purine would be identified as having a guanine base, and so forth.
- FIGURES 1 and 2 provide additional guidance to the reader, but should not be construed as limiting.
- the blocking group will be removed (Step 8b) prior to antibody staining (Step 5).
- the invention is directed to a method of sequencing-by-synthesis (SBS) using unlabeled reversible terminator nucleotides.
- SBS methods are well known including, but not limited to, methods described in references cited herein, each of which is incorporated by reference for all purposes.
- SBS determines sequence of a single-stranded nucleic acid template immobilized at a position on a surface.
- the template copies are most often produced using DNA nanoball (DNB) methods or bridge PCR methods.
- DNS DNA nanoball
- DNB methods result in a single stranded concatemer with many copies of the template (e.g., genomic DNA sequences and adjacent primer binding sites).
- Bridge PCR methods result in a clonal cluster of template molecules (e.g., genomic DNA sequences flanked by adaptors which may serve as primer binding sites). In bridge PCR both strands of the template nucleic acid may be present, as separate single strands.
- templates i.e., singular grammatical form
- array is used in the broadest sense and includes, unless otherwise specified, ordered arrays (meaning template binding regions are arranged in an ordered, typically rectilinear, pattern, such as a grid, spiral, or other patterns) and disordered arrays (meaning template binding regions are at random positions).
- ordered arrays meaning template binding regions are arranged in an ordered, typically rectilinear, pattern, such as a grid, spiral, or other patterns
- disordered arrays meaning template binding regions are at random positions.
- identity of templates at any specific position (or "address”) on an array may be known prior to sequencing of the templates. More often, the array is a "random array” in which the identity of a templates at a given address is not known prior to sequencing.
- array is not limited to positions on a planer surface, but can include bead arrays, droplet arrays, and the like.
- SBS methods can be used with the nucleoside analogues and affinity reagents of the present invention.
- the SBS methods can be selected from those described in U.S. Pat. Nos. 6,210,891; 6,828,100, 6,833,246; 6,911,345; 6,969,488; 6,897,023; 6,833,246; and 6,787,308; U.S. Pat. Pub. Nos. 2003/0064398; 2003/0022207; 2016/0130647; and PCT Pat. Pub. WO 2016/133764; Margulies et al., 2005, Nature 437:376- 380; Ronaghi et al., 1996, Anal. Biochem.
- DNA sequencers that perform sequencing by synthesis are commercially available, for example, from lllumina Inc. (San Diego, CA), including MiniSeq, MiSeq, NextSeq, HiSeq, HiSeq X, and NovaSeq sequencing systems.
- Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product.
- sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, Conn., a Life Technologies subsidiary) or sequencing methods and systems described in U.S. Pat. App. Pub. Nos. 2009/0026082 Al; 2009/0127589 Al; 2010/0137143 Al; or 2010/0282617 Al.
- Another sequencing procedures that use cyclic reactions can be used together with the compositions and methods of the present invention, such as, for example, pyrosequencing.
- Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi et al., Anal Biochem 242:84-89, 1996; Ronaghi, Genome Res. 11:3-11, 2001; Ronaghi et al., Science 281:363, 1998); and U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320.
- PPi inorganic pyrophosphate
- pyrosequencing In pyrosequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected via luciferase-produced photons.
- the sequencing reaction can be monitored via a luminescence detection system.
- Excitation radiation sources used for fluorescence based detection systems are not necessary for pyrosequencing procedures.
- Useful fluidic systems, detectors and procedures that can be used for application of pyrosequencing to arrays of the present disclosure are described, for example, in WIPO Pat. App. Ser. No. PCT/US11/57111, U.S. Pat. App. Pub. No. 2005/0191698 Al, U.S. Pat. No. 7,595,883, and U.S. Pat. No. 7,244,559.
- sequencing by ligation methods can be selected from those described in PCT Pat. Pub. WO 1999/019341; WO 2005/082098; WO 2006/073504; and Shendure et al., 2005, Science, 309: 1728-1739.
- SBS methods can employ the ordered DNA nanoball arrays that are described, for example, in U.S. Pat. Pubs. 2010/0105052, 2007/099208, and US 2009/0264299) and PCT Pat. Pubs. WO 2007/120208, WO 2006/073504, and WO 2007/133831.
- the patent and non-patent publications listed in this paragraph above are hereby incorporated by reference in their entirety for all purposes.
- sequencing is performed on ordered arrays of
- DNBs DNA nanoballs
- DNBs are produced by rolling circle replication of circular library constructs, each containing a fragment of a genome or other target nucleic acid of interest, resulting in a linear single-stranded DNA concatemer comprising multiple copies of the circular construct that collapses in aqueous solution to form a compact ball-like structure.
- the DNBs are disposed on the surface of a two-dimensional planar substrate to form a random array of single molecules.
- DNBs can be fixed directly or indirectly to the surface by a variety of techniques, including covalent attachment and non-covalent attachment.
- patterned substrates with two dimensional arrays of spots are used to produce the DNB array.
- the spots are activated to capture and hold the DNBs, while the DNBs do not remain in the areas between spots.
- a DNB on a spot will repel other DNBs, resulting in one DNB per spot.
- DNBs are three-dimensional (i.e., are not linear short pieces of DNA)
- arrays of the invention result in more DNA copies per square nanometer of binding surface than traditional DNA arrays. This three-dimensional quality further reduces the quantity of sequencing reagents required, resulting in brighter spots and more efficient imaging. Occupancy of DNB arrays often exceed 90%, but can range from 50% to 100% occupancy. Since DNBs are disposed on a surface and then stick to the activated spots in these embodiments, a high-density DNB array essentially "self-assembles" from DNBs in solution.
- the DNB array is contacted with a primer and the primer is extended by one complementary base by a polymerase in each cycle of sequencing.
- the identity of the RT incorporated by the polymerase at each position in the array is revealed as a result of binding of a specific affinity reagent to its corresponding RT.
- the result is an array of DNBs, each of which is labeled with an affinity reagent, such that the identity of the RT incorporated at a particular position on the array is identifiable by the fluorescent label (or other detectable label) that is part of the affinity reagent bound to the RT.
- a template nucleic acid is immobilized on a surface and an oligonucleotide primer is hybridized to a predetermined position on the template (i.e., the primer binding site).
- the incorporated nucleotide analog is complementary to, and basepairs with, the nucleotide at the corresponding position on the template.
- the nucleotide analog includes a detectable label that identifies the nucleobase of the incorporated nucleotide analog, and therefore also identifies the base of the complementary nucleotide in the template.
- the nucleotide analog includes a fluorescent label attached to the nucleobase by a cleavable linker.
- the blocking group is removed, typically chemically or enzymatically, to produce an incorporated nucleotide with a 3' -OH group. Additional rounds of incorporation of 3' blocked nucleotide analogs, detection, and de-blocking may be carried out in additional primer extension reactions.
- extension of the primer although it may be more precise to say that the extension product of the previous round (and not the original oligonucleotide primer) that is extended.
- the primer extension strand may be referred to various ways, including as the "growing DNA strand (GDS)," "primer extension product,” or "extended primer.”
- nucleoside triphosphate i.e., nucleoside triphosphate
- pyrophosphate is removed such that a nucleoside monophosphate (or nucleotide) is incorporated.
- An unlabeled or nonlabeled reversible terminator nucleotide can refer to either form (free nucleoside triphosphate or incorporated nucleotide monophosphate), unless otherwise specified, as will be clear from context.
- An unlabeled, or nonlabeled reversible terminator, nucleotide can be referred to as an NLRT.
- the dNTP analog(s) that is incorporated are not detectably labeled.
- not detectably labeled means that the incorporated dNTP is not conjugated to a dye that produces a detectable (e.g., fluorescent) signal or an enzyme that in the presence of substrates produces a detectable (e.g., chemiluminescent) signal.
- a "reversible terminator nucleotide” refers to a naturally occurring nucleotide, or a nucleotide analog, in which the deoxyribose 3' -OH is replaced with a removable blocking moiety, e.g., 3'-0-azidomethyl.
- NLRT is detected by an affinity reagent(s), such as an antibody(s). , that distinguishes among 3' terminal nucleotides of the primer extension products and thereby identifies the nucleobase of the 3' terminal nucleotide of the template.
- the affinity reagent specifically binds to an incorporated NLRT with containing a specific base (e.g., A, T, G, or C), or analog of the specific base, with much greater affinity than it binds incorporated NLRT with the other bases or other base analogs present in the sequencing reaction.
- the affinity reagent binds to an incorporated NLRT with containing a specific base (e.g., A, T, G, or C), or analog of the specific base, with a characteristic affinity or efficiency that is different from the affinity or efficiency with which it binds to the other bases, or other base analogs present, present in the sequencing reaction.
- a specific base e.g., A, T, G, or C
- the affinity reagents may also distinguish an NLRT incorporated at the 3' terminus of the primer extension product from previously incorporated, "internal,” nucleotides not at the 3' terminus.
- the NLRT at the 3' terminus of the primer extension product differs from previously incorporated nucleotides by the presence of a free 3'-OH (which in internal nucleotides is replaced by a phosphodiester linkage) or the presence of a 3' blocking moiety, as well as differential accessibility of the sugar and nucleobase.
- NLRTs with different nitrogenous bases e.g., A, T, G and C.
- different affinity reagents e.g., 2, 3 or 4 different affinity reagents
- each of which binds an NLRTs with a specific nitrogenous base and does not bind NLRTs with different nitrogenous bases or, in some embodiments, binds NLRTs with different nitrogenous bases or non-identical blocking group but does so at different levels of efficiency.
- the affinity reagent may distinguish one incorporated NLRT from a different
- NLRT based on structural differences in the nitrogenous base, the sugar, the cleavable blocking group or a combination of these elements.
- different NLRTs are distinguished because of, for example, significant structural differences in the nitrogenous base (e.g., adenosine vs guanine) and/or significant structural differences in the blocking group (e.g., azidomethyl vs cyanoethenyl).
- the affinity reagent may distinguish one incorporated NLRT from a different NLRT based on small structural differences (e.g., in some cases, addition or substitution fewer than 5 atoms) preferably in combination with natural differences. These small structural changes can be made in the nitrogenous base, the sugar, and/or blocking group. Affinity reagents such as antibodies can be made that distinguish such small differences between different NLRTs.
- each of the affinity reagents can be distinguished from the other(s) present in the sequencing reaction (for example, because each is differently labeled) or is bound by different secondary binders.
- suitable modified bases will retain normal Watson-Crick binding specificity and should be compatible with incorporation by a DNA polymerase.
- the base analog does not have fluorescent properties (Renatus et al., 2010, Chem Rev. 110(5): 2579-2619).
- the sugar portion of the NLRTs may be modified. Nucleic acids with such modified NLRTs should retain the ability to anneal to the template strand and should be compatible with incorporation by a DNA polymerase.
- NLRT's with blocking groups that differ only slightly may be used. For example, 2, 3, or 4 different such NLRT's may be used.
- a blocking group (not including the deoxyribose 3' oxygen atom) has a molecular weight (MW) less than 184, often less than 174, often less than 164, often less than 154, often less than 144, often less than 134, often less than 124, often less than 114, often less than 104, often less than 94, and sometimes less than 84.
- MW molecular weight
- the molecular weights of deoxyribonucleotide monophosphates are in the range of about 300 to 325 (dAMP 331.2, dCMP 307.2, dGMP 347.2 and dTMP 322.2).
- the NLRT moiety when incorporated into a primer extension product i.e., including the reversible terminator blocking group but not including the pyrophosphate of the dNTP
- the methods of the invention are used to generate sequencing reads longer than 1000 nucleotide, sometimes 10-500 nucleotides, sometimes 10- 250, sometimes more than 25, sometimes more than 50 nucleotides. In some cases sequencing is carried out with fewer than one error per 2000 bases, one error per 5000 bases.
- TSTU (20 mg, 0.066 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, and the desired activated NHS ester A12 was obtained for making biological conjugates.
- T16 Synthesis of the amino-reactive NHS ester of 3'-0-azidomethyl-2'-deoxythymine (T16) is shown in Figure 8.
- Compound T13 108 mg, 0.363 mmol
- anhydrous DMF (1 mL)
- 1,1'- carbonyldiimidazole (CDI) 74 mg, 0.456 mmol
- CDI 1,1'- carbonyldiimidazole
- the reaction mixture was stirred at room temperature for 24 hours.
- Ethyl 4-aminobutyrate hydrochloride (80 mg, 0.477 mmol) and triethylamine (75 uL, 0.539 mmol) were added.
- the mixture was stirred at 40 ' C for 6 hours.
- Most DMF was removed on a rotovap under vacuum to give crude compound T14.
- TSTU 40 mg, 0.132 mmol
- the reaction mixture was stirred at room temperature for 1 hour, and the desired activated NHS ester T16 was obtained for making biological conjugates.
- This example describes protocols use for immunization and antibody purfication use to produce reagents for sequencing. This protocol has been used to make polyclonal antisera with antibodies specific for NLRT- A, -T, -G, and -C with azidomethyl as the 3' blocking group.
- KLH-antigen to inject rabbit
- 3 mg BSA-antigen to titer
- 2-3 ml Sepharose-antigen for purification
- DNA nanoball (DNB) arrays of an f. coli genomic DNA library were used in the sequencing experiments described in the Examples.
- DNBs and DNB arrays are described in, e.g., Drmanac et al., 2010, "Human genome sequencing using unchained base reads on self- assembling DNA nanoarrays," Science 327:78-81, incorporated herein by reference.
- circular library constructs were made from fragments of f. coli genomic DNA, and the library constructs were amplified by rolling circle amplification (RCA) to produce DNBs comprising genomic DNA inserts with adjacent primer binding sites.
- RCA rolling circle amplification
- the DNBs were arrayed in a DNA sequencing flow-cell (e.g., a BGISEQ-500 flow-cell or BGISEQ-1000 flow-cell) and sequencing was carried out using a BGISEQ-500 (BGI, Shenzhen, China; see Huang et al., 2017, "A reference human genome dataset of the BGISEQ-500 sequencer" Gigascience 6:1-9) or a BGISEQ-1000 (BGI, Shenzhen, China).
- a DNA sequencing flow-cell e.g., a BGISEQ-500 flow-cell or BGISEQ-1000 flow-cell
- sequencing was carried out using a BGISEQ-500 (BGI, Shenzhen, China; see Huang et al., 2017, "A reference human genome dataset of the BGISEQ-500 sequencer" Gigascience 6:1-9) or a BGISEQ-1000 (BGI, Shenzhen, China).
- 3'-azidomethyl-dC, 3'-azidomethyl-dG, or 3'-azidomethyl-dT as described in Example 2 were used in this experiment.
- Four (4) different purification preparations of anti-NLRT antbodies were prepared for each of the four bases (i.e., RT-A, RT-C, RT-G and RT-T), resulting in sixteen (16) antibody preparations denoted A1-A4, C1-C4, G1-G4, and T1-T4.
- coli genomic DNA inserts as described in Example 4, were primed and primers were extended using BG9 DNA polymerase (BGI Shenzhen, China), a polymerase eningeered to incorporate 3' modified dNTPs and four non-labeled reversible terminators with a 3'-azidomethyl blocking group (e.g., 3'-azidomethyl-dATP, -dCTP, -dGTP and -dTTP).
- the sixteen (16) antibody preparations were individually applied to speparate lanes on the DNB arrays at 10 ⁇ g/mL and incubated for at 35°C for 5 min (16 separate incubations).
- the highest level of antibody-mediated signal is shown in bold font. Although variation in signal intensity was observed between arrays (depending on the specific preparation of rabbit polyclonal antibody used) the results show that it is possible to meet or exceed the control signal intensity at relatively low antibody concentrations using this indirect detection technique.
- a DNA nanoball £ coli genomic DNA library was sequenced using fluorescently labeled RT-A, -C and -T and unlabeled RT-G, all with 3'-azidomethyl blocking groups. Sequencing was performed using a BGISEQ-500 sequencer (BGI, Shenzhen, China) and data were analyzed using a base calling analysis report provided with the sequencer. Sequencing was carried out for 5 cycles (FIGURES 10A and IOC) or 10 cycles (FIGURES 10B and 10D).
- FIGURES 10A and IOC show the Rho value. (Rho values are calculated by the subtraction of the background from the signal intensities obtained after image analysis. The normalization of the signal is also applied including the cross-talk correction.)
- FIGURES 10B and 10D show the signal-to-noise ratio (SNR).
- the data including the Rho of intensities and SNS (signal-to-noise ratio) from the successful base calling report, indicates that sequencing can be performed successfully with unlabeled RTs and labeled affinity reagents that bind specifically to the RT.
- TABLE 2 illustrates sequencing data generated using a BGISEQ-1000 sequencer
- GDS has 3'-OH rather then an azidomethyl blocking group (Antibody specificity control 2).
- This Example shows results of fifty (50) cycles of sequencing-by-synthesis (SBS) carried out using an BGISEQ-1000 DNA sequencer and an f. coli genomic DNB library.
- a DNA primer complementary to the sequence flanking the genomic DNA insert was hybridized onto the DNB array and primer extension was carried out using 3' -azidomethyl reversible terminators (RT-A, -C, -G, -T) at a concentration of 2 ⁇ each.
- reversible terminators (azidomethyl-A, -C, -T) were fluorescently labeled via cleavable linker attached to the base (used at a ratio of 50% labeled/50% non-labeled) and one reversible terminator (3'- azidomethyl-dGTP) was unlabeled. Primer extension was carried out at 35°C for 2 min using BG9 DNA.
- FIGURE 11A shows the percent Basecall Information Content (BIC). This graph shows that the identity of the unlabeled base when detected indirectly through anti-3'- azidomethyl-dG rabbit primary antibody pre-combined with anti-rabbit AF647 fluorescently labeled fragment secondary antibody provides sufficient information for basecall analysis and identification of the unknown DNA residue(s).
- FIGURE 11B shows the signal intensities and trends for each of the unique fluorescent labels.
- Three nucleotides dATP, dCTP, dTTP
- dATP dATP
- dCTP dCTP
- dTTP fluorescently labeled cleavable linker attached to the base
- Non-labeled 3'-azidomethyl-dGTP was detected by anti-3'-azidomethyl-dG rabbit primary antibody pre-combined with anti-rabbit AF647 fluorescently labeled fragment secondary antibody.
- Control values are results of sequencing using four labeled 3'-azidomethyl RTs
- Pre-stain values are scanned after one round of primer extension but prior to addition of antibody.
- TABLE 3 shows results using polyclonal antibodies against 3'-0-azidomethyl-2'- deoxyadenine.
- TABLE 4 shows results using polyclonal antibodies against 3'-0-azidomethyl-2'- deoxycytosine.
- TABLE 5 shows results using polyclonal antibodies against 3'-0-azidomethyl-2'- deoxyguanine.
- TABLE 6 shows results using polyclonal antibodies against 3'-0-azidomethyl-2'- deoxythymidine.
- E. coli genomic DNA library was made as described in Example 2, and arrayed on a BGISEQ-500 flow-cell. Primers were added and sequencing by synthesis was performed by primer extension using unlabeled nucleotide 3'-azidomethyl reversible terminators (dATP, dCTP, dGTP, dTTP). The unlabeled 3'-blocked dNTPs were present at a concentration of 1 ⁇ each and were incorporated using BG9 DNA at 55°C for 1 min per cycle.
- dATP unlabeled nucleotide 3'-azidomethyl reversible terminators
- the four 3'-azidomethyl-base nucleotides were detected by contacting the array with a mixture of four directly labeled anti-3'-azidomethyl- base antibodies in the concentrations shown inTABLE 11 (range of 10-100 ⁇ g/mL) were incubated on the array at 35oC 2 X 2 min per cycle. "2 x 2" refers to incubation with antibody for two minutes, followed by further 2 minute incubation after adding additional antibody. The array was washed three times to remove any unbound antibodies. TABLE 7 shows shows the identity of fluorophore directly conjugated to each detection antibody.
- the fluorescence signal at each position on the DNB array was determined by scanning for 80 ms during laser excitation of the fluorophore. After DNB base identity was determined, the 3' blocking group was removed by reduction with THPP (13 mM) for 2 min at 35°C, allowing for the regeneration of 3'-OH group and permitting further extension of the nascent DNA strand. Removal of the 3' blocking group also resulted in disassociation of the antibody from the primer extension product.
- FIGURES 12A and 12B show the Rho and signal to noise ratio (SNR) for each base at each sequencing cycle.
- SNR signal to noise ratio
- TABLES 9 and 10 show (Top signal), Rho (background and cross-talk subtracted signal), background signal and signal-to-noise ratio (SNR) values obtained with staining of non- labeled reversible terminators on E. coli genomic DNA using directly labeled anti-azidomethyl- base antibodies.
- This experiment was performed on a BGISEQ-500 flow-cell array with an E. coli genomic DNA library and scanned on a BGISEQ-500 DNA sequencer.
- Primers were hybridized onto the immobilized DNBs and extended in the presence of all four non-labeled nucleotide 3'- azidomethyl RTs (dATP, dCTP, dGTP, dTTP) at 1 ⁇ each concentration for 2 min at 35oC using BG9 polymerase. After incorporation and washing to remove unincorporated nucleotides, the incorporated 3'-azidomethyl-base nucleotides were detected simultaneously by incubating all four labeled anti-3'-azidomethyl-base antibodies (Antibody Set 1) at the concentrations shown (e.g., "@30" means 30 ug/mL) for two sequential incubations of two minutes each at 35°C.
- Antibody Set 1 the concentrations shown (e.g., "@30" means 30 ug/mL) for two sequential incubations of two minutes each at 35°C.
- Antibody Set 2 comprises the same antibody preparations but the antibodies are labeled differently.TABLE 10 shows signal after applying Antibody Set 2. The data demonstrate that signal and SNR values are suitable for basecalling analysis independent of the identity of the directly labeled fluorophore.
- the Example describes a process in which (1) the identity of a base at a first position is determined by detecting the binding by a first primary antibody specific for the base and 3' blocking group; (2) removing the first primary antibody without removing the 3' blocking group; (3) reprobing the same position using a second primary antibody specific for the base and 3' blocking group.
- the results of these experiments are sumarized in TABLE 12.
- TABLE 12 Illustrates an improved DNA sequence identity mapping rate when two independent reads from different fluorescent color combinations (as described in Example 10) are combined for each position of the nascent sequencing-by-synthesis strand for a total of 20 positions read.
- "Odd indep” represents the initial read in “conventional colors” for each sequencing position.
- "Even indep” represents the subsequent read in “alternate colors” after removal by specific competition using the procedure outlined in TABLE 12.
- oCombo represents the result of comparing each of the two independent reads and weighting the result to the higher intensity and therefore higher confidence value of the two reads. Results show significantly higher mapping rates and significantly lower mismatch rates when the two independent reads are combined using this technique.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Priority Applications (16)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR112019013715-7A BR112019013715B1 (en) | 2017-01-04 | 2018-01-04 | METHODS TO DETECT INCORPORATION OF A 3?-O-DEOXYRIBONUCLEOTIDE REVERSIBLE TERMINATOR TO PERFORM A SYNTHESIS SEQUENCING REACTION TO PERFORM DEOXYRIBONUCLEOTIDE SYNTHESIS SEQUENCING AT END 3? OF A STARTER EXTENSION PRODUCT AND FOR NUCLEIC ACID SEQUENCING, DNA ARRANGEMENT, E, KIT |
RS20220695A RS63419B1 (en) | 2017-01-04 | 2018-01-04 | Nucleic acid sequencing using affinity reagents |
CN201880005910.5A CN110418847A (en) | 2017-01-04 | 2018-01-04 | Pass through the substep sequencing of the reversible terminator or natural nucleotide of non-marked |
EP22174722.3A EP4112741A1 (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides |
EP18736171.2A EP3565905B1 (en) | 2017-01-04 | 2018-01-04 | Nucleic acid sequencing using affinity reagents |
KR1020197022620A KR102349259B1 (en) | 2017-01-04 | 2018-01-04 | Nucleic Acid Sequencing Using Affinity Reagents |
MX2019008016A MX2019008016A (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides. |
DK18736171.2T DK3565905T3 (en) | 2017-01-04 | 2018-01-04 | NUCLEIC ACID SEQUENCE USING AFFINITY REAGENTS |
CA3048486A CA3048486A1 (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides |
ES18736171T ES2924223T3 (en) | 2017-01-04 | 2018-01-04 | Nucleic acid sequencing using affinity reagents |
JP2019536551A JP2020508037A (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing with unlabeled reversible terminators or natural nucleotides |
IL267836A IL267836B2 (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides |
PL18736171.2T PL3565905T3 (en) | 2017-01-04 | 2018-01-04 | Nucleic acid sequencing using affinity reagents |
AU2018205472A AU2018205472B2 (en) | 2017-01-04 | 2018-01-04 | Nucleic acid sequencing using affinity reagents |
JP2021212172A JP7025595B1 (en) | 2017-01-04 | 2021-12-27 | Nucleic acid sequencing using affinity reagents |
AU2023201547A AU2023201547A1 (en) | 2017-01-04 | 2023-03-14 | Nucleic acid sequencing using affinity reagents |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762442263P | 2017-01-04 | 2017-01-04 | |
US62/442,263 | 2017-01-04 | ||
US201762490511P | 2017-04-26 | 2017-04-26 | |
US62/490,511 | 2017-04-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018129214A1 true WO2018129214A1 (en) | 2018-07-12 |
Family
ID=62791177
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2018/012425 WO2018129214A1 (en) | 2017-01-04 | 2018-01-04 | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides |
Country Status (17)
Country | Link |
---|---|
US (3) | US10851410B2 (en) |
EP (2) | EP3565905B1 (en) |
JP (2) | JP2020508037A (en) |
KR (1) | KR102349259B1 (en) |
CN (1) | CN110418847A (en) |
AU (2) | AU2018205472B2 (en) |
BR (1) | BR112019013715B1 (en) |
CA (1) | CA3048486A1 (en) |
DK (1) | DK3565905T3 (en) |
ES (1) | ES2924223T3 (en) |
HU (1) | HUE059673T2 (en) |
IL (1) | IL267836B2 (en) |
MX (2) | MX2019008016A (en) |
PL (1) | PL3565905T3 (en) |
PT (1) | PT3565905T (en) |
RS (1) | RS63419B1 (en) |
WO (1) | WO2018129214A1 (en) |
Cited By (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020097607A1 (en) * | 2018-11-09 | 2020-05-14 | Mgi Tech Co., Ltd. | Massively parallel sequencing using unlabeled nucleotides |
EP3702474A1 (en) * | 2019-02-26 | 2020-09-02 | QIAGEN GmbH | Sequencing method and kit |
US10851410B2 (en) | 2017-01-04 | 2020-12-01 | Mgi Tech Co., Ltd. | Nucleic acid sequencing using affinity reagents |
CN112840035A (en) * | 2018-11-07 | 2021-05-25 | 深圳华大智造极创科技有限公司 | Method for sequencing polynucleotides |
JP2022511546A (en) * | 2018-12-05 | 2022-01-31 | 深▲せん▼華大智造極創科技有限公司 | Rolling circle amplification method, sequencing library preparation method and prepared DNA nanospheres |
WO2022023353A1 (en) | 2020-07-28 | 2022-02-03 | Illumina Cambridge Limited | Substituted coumarin dyes and uses as fluorescent labels |
WO2022078319A1 (en) * | 2020-10-12 | 2022-04-21 | Mgi Tech Co., Ltd. | Protein detection and tracking using nucleoside tags |
WO2022079005A1 (en) * | 2020-10-12 | 2022-04-21 | Cambridge Display Technology Limited | Method comprising light emitting marker |
WO2022083686A1 (en) | 2020-10-21 | 2022-04-28 | 深圳华大生命科学研究院 | Modified nucleoside or nucleotide |
WO2022129930A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Alkylpyridinium coumarin dyes and uses in sequencing applications |
WO2022129439A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Methods, systems and compositions for nucleic acid sequencing |
WO2022129437A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Long stokes shift chromenoquinoline dyes and uses in sequencing applications |
WO2022136402A1 (en) * | 2020-12-22 | 2022-06-30 | Illumina Cambridge Limited | Methods and compositions for nucleic acid sequencing |
WO2022206922A1 (en) | 2021-04-01 | 2022-10-06 | 深圳华大生命科学研究院 | Nucleotide analogue for sequencing |
WO2022233795A1 (en) | 2021-05-05 | 2022-11-10 | Illumina Cambridge Limited | Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing |
JP2023506891A (en) * | 2019-12-18 | 2023-02-20 | エフ. ホフマン-ラ ロシュ エージー. | Sequencing-by-synthesis methods using sequential labeling schemes |
WO2023114896A1 (en) | 2021-12-16 | 2023-06-22 | Illumina Cambridge Limited | Methods for metal directed cleavage of surface-bound polynucleotides |
WO2023122499A1 (en) | 2021-12-20 | 2023-06-29 | Illumina Cambridge Limited | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides |
WO2023122491A1 (en) | 2021-12-20 | 2023-06-29 | Illumina Cambridge Limited | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides |
WO2023186819A1 (en) | 2022-03-29 | 2023-10-05 | Illumina Cambridge Limited | Chromenoquinoline dyes and uses in sequencing |
WO2023186872A1 (en) | 2022-03-30 | 2023-10-05 | Illumina Cambridge Limited | Methods for chemical cleavage of surface-bound polynucleotides |
WO2023192900A1 (en) | 2022-03-31 | 2023-10-05 | Illumina Singapore Pte. Ltd. | Nucleosides and nucleotides with 3' vinyl blocking group useful in sequencing by synthesis |
WO2023186982A1 (en) | 2022-03-31 | 2023-10-05 | Illumina, Inc. | Compositions and methods for improving sequencing signals |
WO2024003087A1 (en) | 2022-06-28 | 2024-01-04 | Illumina, Inc. | Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing |
WO2024068889A2 (en) | 2022-09-30 | 2024-04-04 | Illumina, Inc. | Compositions and methods for reducing photo damage during sequencing |
WO2024123866A1 (en) | 2022-12-09 | 2024-06-13 | Illumina, Inc. | Nucleosides and nucleotides with 3´ blocking groups and cleavable linkers |
WO2024137765A1 (en) | 2022-12-22 | 2024-06-27 | Illumina, Inc. | Transition-metal catalyst compositions and methods for sequencing by synthesis |
WO2024137774A1 (en) | 2022-12-22 | 2024-06-27 | Illumina, Inc. | Palladium catalyst compositions and methods for sequencing by synthesis |
WO2024145154A1 (en) | 2022-12-27 | 2024-07-04 | Illumina, Inc. | Methods of sequencing using 3´ allyl blocked nucleotides |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019067635A1 (en) | 2017-09-27 | 2019-04-04 | Ultima Genomics, Inc. | Methods and systems for nucleic acid sequencing |
JP7485483B2 (en) * | 2019-05-15 | 2024-05-16 | チンタオ エムジーアイ テック カンパニー リミテッド | A single-channel sequencing method based on autoluminescence |
US20220145379A1 (en) * | 2019-12-16 | 2022-05-12 | Illumina, Inc. | Kits and flow cells |
US20210189483A1 (en) | 2019-12-23 | 2021-06-24 | Mgi Tech Co. Ltd. | Controlled strand-displacement for paired end sequencing |
CN115427586A (en) | 2020-03-18 | 2022-12-02 | 深圳华大智造科技股份有限公司 | Recovery phase in massively parallel sequencing |
US11761040B2 (en) | 2020-04-20 | 2023-09-19 | Mgi Tech Co., Ltd. | DNA protection agent in DNA imaging buffer |
WO2021222512A1 (en) | 2020-04-30 | 2021-11-04 | Dimensiongen | Devices and methods for macromolecular manipulation |
WO2022197589A1 (en) | 2021-03-15 | 2022-09-22 | President And Fellows Of Harvard College | Methods for in situ sequencing |
CN113372402A (en) * | 2021-06-15 | 2021-09-10 | 中国人民解放军军事科学院军事医学研究院 | 3' -O-reversibly blocked nucleotides and their use in template-free enzymatic nucleic acid synthesis |
CN116536399A (en) * | 2023-07-07 | 2023-08-04 | 之江实验室 | Gene chip coding and decoding method based on microbead sequencing and gene chip |
CN118496347A (en) * | 2024-05-30 | 2024-08-16 | 北京航空航天大学杭州创新研究院 | Method for coupling antibody oligonucleotides |
Citations (75)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4351760A (en) | 1979-09-07 | 1982-09-28 | Syva Company | Novel alkyl substituted fluorescent compounds and polyamino acid conjugates |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4800159A (en) | 1986-02-07 | 1989-01-24 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences |
US4965188A (en) | 1986-08-22 | 1990-10-23 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme |
US5188934A (en) | 1989-11-14 | 1993-02-23 | Applied Biosystems, Inc. | 4,7-dichlorofluorescein dyes as molecular probes |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US5399491A (en) | 1989-07-11 | 1995-03-21 | Gen-Probe Incorporated | Nucleic acid sequence amplification methods |
US5854033A (en) | 1995-11-21 | 1998-12-29 | Yale University | Rolling circle replication reporter systems |
WO1999019341A1 (en) | 1997-10-10 | 1999-04-22 | President & Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
US6174670B1 (en) | 1996-06-04 | 2001-01-16 | University Of Utah Research Foundation | Monitoring amplification of DNA during PCR |
US6210891B1 (en) | 1996-09-27 | 2001-04-03 | Pyrosequencing Ab | Method of sequencing DNA |
US6232465B1 (en) | 1994-09-02 | 2001-05-15 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides |
US6258568B1 (en) | 1996-12-23 | 2001-07-10 | Pyrosequencing Ab | Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation |
US6274320B1 (en) | 1999-09-16 | 2001-08-14 | Curagen Corporation | Method of sequencing a nucleic acid |
US6287824B1 (en) | 1998-09-15 | 2001-09-11 | Yale University | Molecular cloning using rolling circle amplification |
US20020027131A1 (en) | 2000-09-01 | 2002-03-07 | Makoto Kitada | Thermal control apparatus and method for thermally controlling a plurality of objects within a predetermined temperature range |
US6396995B1 (en) | 1999-05-20 | 2002-05-28 | Illumina, Inc. | Method and apparatus for retaining and presenting at least one microsphere array to solutions and/or to optical imaging systems |
US6401267B1 (en) | 1993-09-27 | 2002-06-11 | Radoje Drmanac | Methods and compositions for efficient nucleic acid sequencing |
WO2002103039A1 (en) * | 2001-06-14 | 2002-12-27 | Cavidi Tech Ab | A method for measuring dna polymerization and applications of the method |
WO2003003014A1 (en) * | 2001-06-29 | 2003-01-09 | Veri-Q, Inc. | Methods and compositions for determining the purity of and purifying chemically synthesized nucleic acids |
US20030022207A1 (en) | 1998-10-16 | 2003-01-30 | Solexa, Ltd. | Arrayed polynucleotides and their use in genome analysis |
US20030064398A1 (en) | 2000-02-02 | 2003-04-03 | Solexa, Ltd. | Synthesis of spatially addressed molecular arrays |
US6544732B1 (en) | 1999-05-20 | 2003-04-08 | Illumina, Inc. | Encoding and decoding of array sensors utilizing nanocrystals |
WO2003073088A2 (en) | 2001-03-09 | 2003-09-04 | Christofer Toumazou | Apparatus and method for the detection of localised fluctuactions of ionic charge by ion sensitive field effect transistors during a chemical reaction |
US6787308B2 (en) | 1998-07-30 | 2004-09-07 | Solexa Ltd. | Arrayed biomolecules and their use in sequencing |
US6828100B1 (en) | 1999-01-22 | 2004-12-07 | Biotage Ab | Method of DNA sequencing |
US6833246B2 (en) | 1999-09-29 | 2004-12-21 | Solexa, Ltd. | Polynucleotide sequencing |
WO2005024010A1 (en) | 2003-09-11 | 2005-03-17 | Solexa Limited | Modified polymerases for improved incorporation of nucleotide analogues |
US6897023B2 (en) | 2000-09-27 | 2005-05-24 | The Molecular Sciences Institute, Inc. | Method for determining relative abundance of nucleic acid sequences |
US6911345B2 (en) | 1999-06-28 | 2005-06-28 | California Institute Of Technology | Methods and apparatus for analyzing polynucleotide sequences |
US20050191698A1 (en) | 1999-04-20 | 2005-09-01 | Illumina, Inc. | Nucleic acid sequencing using microsphere arrays |
WO2005082098A2 (en) | 2004-02-27 | 2005-09-09 | President And Fellows Of Harvard College | Polony fluorescent in situ sequencing beads |
US6969488B2 (en) | 1998-05-22 | 2005-11-29 | Solexa, Inc. | System and apparatus for sequential processing of analytes |
US20060024711A1 (en) | 2004-07-02 | 2006-02-02 | Helicos Biosciences Corporation | Methods for nucleic acid amplification and sequence determination |
WO2006073504A2 (en) | 2004-08-04 | 2006-07-13 | President And Fellows Of Harvard College | Wobble sequencing |
US7115400B1 (en) | 1998-09-30 | 2006-10-03 | Solexa Ltd. | Methods of nucleic acid amplification and sequencing |
WO2006120433A1 (en) | 2005-05-10 | 2006-11-16 | Solexa Limited | Improved polymerases |
US20070099208A1 (en) | 2005-06-15 | 2007-05-03 | Radoje Drmanac | Single molecule arrays for genetic and chemical analysis |
US7244559B2 (en) | 1999-09-16 | 2007-07-17 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
WO2007120208A2 (en) | 2005-11-14 | 2007-10-25 | President And Fellows Of Harvard College | Nanogrid rolling circle dna sequencing |
WO2007133831A2 (en) | 2006-02-24 | 2007-11-22 | Callida Genomics, Inc. | High throughput genome sequencing on dna arrays |
US20090026082A1 (en) | 2006-12-14 | 2009-01-29 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes using large scale FET arrays |
US20090127589A1 (en) | 2006-12-14 | 2009-05-21 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes using large scale FET arrays |
US7595883B1 (en) | 2002-09-16 | 2009-09-29 | The Board Of Trustees Of The Leland Stanford Junior University | Biological analysis arrangement and approach therefor |
US20090264299A1 (en) | 2006-02-24 | 2009-10-22 | Complete Genomics, Inc. | High throughput genome sequencing on DNA arrays |
WO2009136182A1 (en) | 2008-04-18 | 2009-11-12 | University Of Leeds | Modified stefin a scaffold proteins |
US20100105052A1 (en) | 2007-10-29 | 2010-04-29 | Complete Genomics, Inc. | Nucleic acid sequencing and process |
US20100137143A1 (en) | 2008-10-22 | 2010-06-03 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes |
US20100282617A1 (en) | 2006-12-14 | 2010-11-11 | Ion Torrent Systems Incorporated | Methods and apparatus for detecting molecular interactions using fet arrays |
US20100311602A1 (en) | 2006-10-13 | 2010-12-09 | J. Craig Venter Institute, Inc. | Sequencing method |
US7897344B2 (en) | 2007-11-06 | 2011-03-01 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs |
US7901890B2 (en) | 2007-11-05 | 2011-03-08 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors employing selective methylation |
US7906285B2 (en) | 2003-02-26 | 2011-03-15 | Callida Genomics, Inc. | Random array DNA analysis by hybridization |
US7910302B2 (en) | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
US7960104B2 (en) | 2005-10-07 | 2011-06-14 | Callida Genomics, Inc. | Self-assembled single molecule arrays and uses thereof |
US8063019B2 (en) | 2005-06-10 | 2011-11-22 | Medical Research Council | Scaffold polypeptides for heterologous peptide display |
US8236499B2 (en) | 2008-03-28 | 2012-08-07 | Pacific Biosciences Of California, Inc. | Methods and compositions for nucleic acid sample preparation |
US8298768B2 (en) | 2007-11-29 | 2012-10-30 | Complete Genomics, Inc. | Efficient shotgun sequencing methods |
WO2013044018A1 (en) * | 2011-09-23 | 2013-03-28 | Illumina, Inc. | Methods and compositions for nucleic acid sequencing |
US8415099B2 (en) | 2007-11-05 | 2013-04-09 | Complete Genomics, Inc. | Efficient base determination in sequencing reactions |
US20130338008A1 (en) | 2008-12-23 | 2013-12-19 | Illumina, Inc. | Method of making an array of nucleic acid colonies |
US8617811B2 (en) | 2008-01-28 | 2013-12-31 | Complete Genomics, Inc. | Methods and compositions for efficient base calling in sequencing reactions |
US8671811B2 (en) | 2011-09-30 | 2014-03-18 | Robert Bosch Gmbh | Pivoting saw system |
WO2014114665A1 (en) | 2013-01-23 | 2014-07-31 | Dynamic Biosensors Gmbh | Method for sequencing a template nucleic acid immobilized on a substrate |
US20140228223A1 (en) | 2010-05-10 | 2014-08-14 | Andreas Gnirke | High throughput paired-end sequencing of large-insert clone libraries |
US20140242579A1 (en) * | 2013-02-28 | 2014-08-28 | Centrillion Technology Holdings Corporation | Reversible terminator molecules and methods of their use |
US9005565B2 (en) | 2010-06-24 | 2015-04-14 | Hamid-Reza Jahangiri-Famenini | Method and apparatus for forming graphene |
US20150353926A1 (en) | 2014-01-16 | 2015-12-10 | Illumina Cambridge Limited | Polynucleotide modification on solid support |
WO2016065248A1 (en) * | 2014-10-23 | 2016-04-28 | Complete Genomics, Inc. | Signal confinement sequencing (scs) and nucleotide analogues for signal confinement sequencing |
WO2016133764A1 (en) | 2015-02-17 | 2016-08-25 | Complete Genomics, Inc. | Dna sequencing using controlled strand displacement |
US9702001B2 (en) | 2010-08-25 | 2017-07-11 | Pacific Biosciences Of California, Inc. | Scaffold-based polymerase enzyme substrates |
US20170240961A1 (en) | 2016-02-23 | 2017-08-24 | Complete Genomics, Inc. | Affinity tag labeled nucleosides and uses |
WO2017185026A1 (en) * | 2016-04-22 | 2017-10-26 | Complete Genomics, Inc. | Reversibly blocked nucleoside analogues and their use |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2713102A (en) | 1952-06-25 | 1955-07-12 | Railroad Accessories Corp | Test terminal |
US5053336A (en) * | 1989-07-17 | 1991-10-01 | Regents Of The University Of California | Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use |
US6743605B1 (en) | 1998-06-24 | 2004-06-01 | Enzo Life Sciences, Inc. | Linear amplification of specific nucleic acid sequences |
JP2001245698A (en) * | 1999-11-22 | 2001-09-11 | Xiao Bing Wang | Method for detecting nucleic acid |
ATE356222T1 (en) | 2000-10-06 | 2007-03-15 | Univ Columbia | MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA |
US20030124560A1 (en) | 2001-06-14 | 2003-07-03 | Yoshihiko Makino | Method for analyzing a target nucleic acid fragment and a kit for analyzing a target nucleic acid fragment |
FR2836995B1 (en) * | 2002-03-05 | 2006-09-22 | Centre Nat Rech Scient | USE OF NUCLEOSIDE DERIVATIVES COMPRISING A CITRATE GROUP FOR THE PRODUCTION OF ANTIBODIES HAVING AFFINITY FOR TRIPHOSPHORYLATED NUCLEOSIDES AND THEIR APPLICATIONS |
WO2005003375A2 (en) | 2003-01-29 | 2005-01-13 | 454 Corporation | Methods of amplifying and sequencing nucleic acids |
WO2004074503A2 (en) | 2003-02-21 | 2004-09-02 | Hoser Mark J | Nucleic acid sequencing methods, kits and reagents |
CN104263816B (en) * | 2008-05-16 | 2018-10-19 | 生命技术公司 | Double label method for measuring cell Proliferation |
CN104448001A (en) | 2014-11-14 | 2015-03-25 | 杭州博谱医药科技有限公司 | Anti-5-methylcytosine monoclonal antibody and application thereof |
AU2017313718B2 (en) | 2016-08-15 | 2023-09-14 | Pacific Biosciences Of California, Inc. | Method and system for sequencing nucleic acids |
WO2018129214A1 (en) | 2017-01-04 | 2018-07-12 | Complete Genomics, Inc. | Stepwise sequencing by non-labeled reversible terminators or natural nucleotides |
-
2018
- 2018-01-04 WO PCT/US2018/012425 patent/WO2018129214A1/en unknown
- 2018-01-04 KR KR1020197022620A patent/KR102349259B1/en active IP Right Review Request
- 2018-01-04 US US15/862,566 patent/US10851410B2/en active Active
- 2018-01-04 MX MX2019008016A patent/MX2019008016A/en unknown
- 2018-01-04 AU AU2018205472A patent/AU2018205472B2/en active Active
- 2018-01-04 DK DK18736171.2T patent/DK3565905T3/en active
- 2018-01-04 EP EP18736171.2A patent/EP3565905B1/en active Active
- 2018-01-04 PL PL18736171.2T patent/PL3565905T3/en unknown
- 2018-01-04 RS RS20220695A patent/RS63419B1/en unknown
- 2018-01-04 ES ES18736171T patent/ES2924223T3/en active Active
- 2018-01-04 HU HUE18736171A patent/HUE059673T2/en unknown
- 2018-01-04 BR BR112019013715-7A patent/BR112019013715B1/en active IP Right Grant
- 2018-01-04 CN CN201880005910.5A patent/CN110418847A/en active Pending
- 2018-01-04 PT PT187361712T patent/PT3565905T/en unknown
- 2018-01-04 IL IL267836A patent/IL267836B2/en unknown
- 2018-01-04 EP EP22174722.3A patent/EP4112741A1/en active Pending
- 2018-01-04 JP JP2019536551A patent/JP2020508037A/en not_active Withdrawn
- 2018-01-04 CA CA3048486A patent/CA3048486A1/en active Pending
-
2019
- 2019-07-03 MX MX2024006412A patent/MX2024006412A/en unknown
-
2020
- 2020-10-29 US US17/084,482 patent/US11788138B2/en active Active
-
2021
- 2021-12-27 JP JP2021212172A patent/JP7025595B1/en active Active
-
2023
- 2023-03-14 AU AU2023201547A patent/AU2023201547A1/en active Pending
- 2023-09-07 US US18/462,652 patent/US20240117425A1/en active Pending
Patent Citations (93)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4351760A (en) | 1979-09-07 | 1982-09-28 | Syva Company | Novel alkyl substituted fluorescent compounds and polyamino acid conjugates |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4683202B1 (en) | 1985-03-28 | 1990-11-27 | Cetus Corp | |
US4683195A (en) | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683195B1 (en) | 1986-01-30 | 1990-11-27 | Cetus Corp | |
US4800159A (en) | 1986-02-07 | 1989-01-24 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences |
US4965188A (en) | 1986-08-22 | 1990-10-23 | Cetus Corporation | Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme |
US5399491A (en) | 1989-07-11 | 1995-03-21 | Gen-Probe Incorporated | Nucleic acid sequence amplification methods |
US5188934A (en) | 1989-11-14 | 1993-02-23 | Applied Biosystems, Inc. | 4,7-dichlorofluorescein dyes as molecular probes |
US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US7070927B2 (en) | 1993-09-27 | 2006-07-04 | University Of Chicago | Methods and compositions for efficient nucleic acid sequencing |
US6401267B1 (en) | 1993-09-27 | 2002-06-11 | Radoje Drmanac | Methods and compositions for efficient nucleic acid sequencing |
US6232465B1 (en) | 1994-09-02 | 2001-05-15 | Andrew C. Hiatt | Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides |
US5854033A (en) | 1995-11-21 | 1998-12-29 | Yale University | Rolling circle replication reporter systems |
US6174670B1 (en) | 1996-06-04 | 2001-01-16 | University Of Utah Research Foundation | Monitoring amplification of DNA during PCR |
US6210891B1 (en) | 1996-09-27 | 2001-04-03 | Pyrosequencing Ab | Method of sequencing DNA |
US6258568B1 (en) | 1996-12-23 | 2001-07-10 | Pyrosequencing Ab | Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation |
WO1999019341A1 (en) | 1997-10-10 | 1999-04-22 | President & Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
US6969488B2 (en) | 1998-05-22 | 2005-11-29 | Solexa, Inc. | System and apparatus for sequential processing of analytes |
US6787308B2 (en) | 1998-07-30 | 2004-09-07 | Solexa Ltd. | Arrayed biomolecules and their use in sequencing |
US6287824B1 (en) | 1998-09-15 | 2001-09-11 | Yale University | Molecular cloning using rolling circle amplification |
US7115400B1 (en) | 1998-09-30 | 2006-10-03 | Solexa Ltd. | Methods of nucleic acid amplification and sequencing |
US20030022207A1 (en) | 1998-10-16 | 2003-01-30 | Solexa, Ltd. | Arrayed polynucleotides and their use in genome analysis |
US6828100B1 (en) | 1999-01-22 | 2004-12-07 | Biotage Ab | Method of DNA sequencing |
US20050191698A1 (en) | 1999-04-20 | 2005-09-01 | Illumina, Inc. | Nucleic acid sequencing using microsphere arrays |
US6544732B1 (en) | 1999-05-20 | 2003-04-08 | Illumina, Inc. | Encoding and decoding of array sensors utilizing nanocrystals |
US6396995B1 (en) | 1999-05-20 | 2002-05-28 | Illumina, Inc. | Method and apparatus for retaining and presenting at least one microsphere array to solutions and/or to optical imaging systems |
US6911345B2 (en) | 1999-06-28 | 2005-06-28 | California Institute Of Technology | Methods and apparatus for analyzing polynucleotide sequences |
US6274320B1 (en) | 1999-09-16 | 2001-08-14 | Curagen Corporation | Method of sequencing a nucleic acid |
US7244559B2 (en) | 1999-09-16 | 2007-07-17 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
US6833246B2 (en) | 1999-09-29 | 2004-12-21 | Solexa, Ltd. | Polynucleotide sequencing |
US20030064398A1 (en) | 2000-02-02 | 2003-04-03 | Solexa, Ltd. | Synthesis of spatially addressed molecular arrays |
US20020027131A1 (en) | 2000-09-01 | 2002-03-07 | Makoto Kitada | Thermal control apparatus and method for thermally controlling a plurality of objects within a predetermined temperature range |
US6897023B2 (en) | 2000-09-27 | 2005-05-24 | The Molecular Sciences Institute, Inc. | Method for determining relative abundance of nucleic acid sequences |
WO2003073088A2 (en) | 2001-03-09 | 2003-09-04 | Christofer Toumazou | Apparatus and method for the detection of localised fluctuactions of ionic charge by ion sensitive field effect transistors during a chemical reaction |
WO2002103039A1 (en) * | 2001-06-14 | 2002-12-27 | Cavidi Tech Ab | A method for measuring dna polymerization and applications of the method |
WO2003003014A1 (en) * | 2001-06-29 | 2003-01-09 | Veri-Q, Inc. | Methods and compositions for determining the purity of and purifying chemically synthesized nucleic acids |
US7595883B1 (en) | 2002-09-16 | 2009-09-29 | The Board Of Trustees Of The Leland Stanford Junior University | Biological analysis arrangement and approach therefor |
US7910304B2 (en) | 2003-02-26 | 2011-03-22 | Callida Genomics, Inc. | Random array DNA analysis by hybridization |
US8105771B2 (en) | 2003-02-26 | 2012-01-31 | Callida Genomics, Inc. | Random array DNA analysis by hybridization |
US7906285B2 (en) | 2003-02-26 | 2011-03-15 | Callida Genomics, Inc. | Random array DNA analysis by hybridization |
US8278039B2 (en) | 2003-02-26 | 2012-10-02 | Complete Genomics, Inc. | Random array DNA analysis by hybridization |
WO2005024010A1 (en) | 2003-09-11 | 2005-03-17 | Solexa Limited | Modified polymerases for improved incorporation of nucleotide analogues |
US20070087362A1 (en) | 2004-02-27 | 2007-04-19 | President And Fellows Of Harvard College | Polony fluorescent in situ sequencing beads |
WO2005082098A2 (en) | 2004-02-27 | 2005-09-09 | President And Fellows Of Harvard College | Polony fluorescent in situ sequencing beads |
US20060024711A1 (en) | 2004-07-02 | 2006-02-02 | Helicos Biosciences Corporation | Methods for nucleic acid amplification and sequence determination |
US20070207482A1 (en) | 2004-08-04 | 2007-09-06 | President And Fellows Of Harvard College | Wobble sequencing |
WO2006073504A2 (en) | 2004-08-04 | 2006-07-13 | President And Fellows Of Harvard College | Wobble sequencing |
WO2006120433A1 (en) | 2005-05-10 | 2006-11-16 | Solexa Limited | Improved polymerases |
US8481491B2 (en) | 2005-06-10 | 2013-07-09 | Medical Research Council | Scaffold polypeptides for heterologous peptide display |
US8063019B2 (en) | 2005-06-10 | 2011-11-22 | Medical Research Council | Scaffold polypeptides for heterologous peptide display |
US8133719B2 (en) | 2005-06-15 | 2012-03-13 | Callida Genomics, Inc. | Methods for making single molecule arrays |
US7901891B2 (en) | 2005-06-15 | 2011-03-08 | Callida Genomics, Inc. | Nucleic acid analysis by random mixtures of non-overlapping fragments |
US7709197B2 (en) | 2005-06-15 | 2010-05-04 | Callida Genomics, Inc. | Nucleic acid analysis by random mixtures of non-overlapping fragments |
US20070099208A1 (en) | 2005-06-15 | 2007-05-03 | Radoje Drmanac | Single molecule arrays for genetic and chemical analysis |
US8445197B2 (en) | 2005-06-15 | 2013-05-21 | Callida Genomics, Inc. | Single molecule arrays for genetic and chemical analysis |
US8445194B2 (en) | 2005-06-15 | 2013-05-21 | Callida Genomics, Inc. | Single molecule arrays for genetic and chemical analysis |
US8445196B2 (en) | 2005-06-15 | 2013-05-21 | Callida Genomics, Inc. | Single molecule arrays for genetic and chemical analysis |
US7960104B2 (en) | 2005-10-07 | 2011-06-14 | Callida Genomics, Inc. | Self-assembled single molecule arrays and uses thereof |
WO2007120208A2 (en) | 2005-11-14 | 2007-10-25 | President And Fellows Of Harvard College | Nanogrid rolling circle dna sequencing |
US20090264299A1 (en) | 2006-02-24 | 2009-10-22 | Complete Genomics, Inc. | High throughput genome sequencing on DNA arrays |
WO2007133831A2 (en) | 2006-02-24 | 2007-11-22 | Callida Genomics, Inc. | High throughput genome sequencing on dna arrays |
US8440397B2 (en) | 2006-02-24 | 2013-05-14 | Callida Genomics, Inc. | High throughput genome sequencing on DNA arrays |
US20100311602A1 (en) | 2006-10-13 | 2010-12-09 | J. Craig Venter Institute, Inc. | Sequencing method |
US7910302B2 (en) | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
US7910354B2 (en) | 2006-10-27 | 2011-03-22 | Complete Genomics, Inc. | Efficient arrays of amplified polynucleotides |
US20100282617A1 (en) | 2006-12-14 | 2010-11-11 | Ion Torrent Systems Incorporated | Methods and apparatus for detecting molecular interactions using fet arrays |
US20090127589A1 (en) | 2006-12-14 | 2009-05-21 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes using large scale FET arrays |
US20090026082A1 (en) | 2006-12-14 | 2009-01-29 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes using large scale FET arrays |
US20100105052A1 (en) | 2007-10-29 | 2010-04-29 | Complete Genomics, Inc. | Nucleic acid sequencing and process |
US8415099B2 (en) | 2007-11-05 | 2013-04-09 | Complete Genomics, Inc. | Efficient base determination in sequencing reactions |
US7901890B2 (en) | 2007-11-05 | 2011-03-08 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors employing selective methylation |
US7897344B2 (en) | 2007-11-06 | 2011-03-01 | Complete Genomics, Inc. | Methods and oligonucleotide designs for insertion of multiple adaptors into library constructs |
US8298768B2 (en) | 2007-11-29 | 2012-10-30 | Complete Genomics, Inc. | Efficient shotgun sequencing methods |
US8617811B2 (en) | 2008-01-28 | 2013-12-31 | Complete Genomics, Inc. | Methods and compositions for efficient base calling in sequencing reactions |
US8236499B2 (en) | 2008-03-28 | 2012-08-07 | Pacific Biosciences Of California, Inc. | Methods and compositions for nucleic acid sample preparation |
WO2009136182A1 (en) | 2008-04-18 | 2009-11-12 | University Of Leeds | Modified stefin a scaffold proteins |
US20100137143A1 (en) | 2008-10-22 | 2010-06-03 | Ion Torrent Systems Incorporated | Methods and apparatus for measuring analytes |
US20130338008A1 (en) | 2008-12-23 | 2013-12-19 | Illumina, Inc. | Method of making an array of nucleic acid colonies |
US20140228223A1 (en) | 2010-05-10 | 2014-08-14 | Andreas Gnirke | High throughput paired-end sequencing of large-insert clone libraries |
US9005565B2 (en) | 2010-06-24 | 2015-04-14 | Hamid-Reza Jahangiri-Famenini | Method and apparatus for forming graphene |
US9702001B2 (en) | 2010-08-25 | 2017-07-11 | Pacific Biosciences Of California, Inc. | Scaffold-based polymerase enzyme substrates |
WO2013044018A1 (en) * | 2011-09-23 | 2013-03-28 | Illumina, Inc. | Methods and compositions for nucleic acid sequencing |
US8671811B2 (en) | 2011-09-30 | 2014-03-18 | Robert Bosch Gmbh | Pivoting saw system |
WO2014114665A1 (en) | 2013-01-23 | 2014-07-31 | Dynamic Biosensors Gmbh | Method for sequencing a template nucleic acid immobilized on a substrate |
US20140242579A1 (en) * | 2013-02-28 | 2014-08-28 | Centrillion Technology Holdings Corporation | Reversible terminator molecules and methods of their use |
US20150353926A1 (en) | 2014-01-16 | 2015-12-10 | Illumina Cambridge Limited | Polynucleotide modification on solid support |
WO2016065248A1 (en) * | 2014-10-23 | 2016-04-28 | Complete Genomics, Inc. | Signal confinement sequencing (scs) and nucleotide analogues for signal confinement sequencing |
US20160130647A1 (en) | 2014-10-23 | 2016-05-12 | Complete Genomics, Inc. | Signal confinement sequencing (scs) and nucleotide analogues for signal confinement sequencing |
WO2016133764A1 (en) | 2015-02-17 | 2016-08-25 | Complete Genomics, Inc. | Dna sequencing using controlled strand displacement |
US20170240961A1 (en) | 2016-02-23 | 2017-08-24 | Complete Genomics, Inc. | Affinity tag labeled nucleosides and uses |
WO2017185026A1 (en) * | 2016-04-22 | 2017-10-26 | Complete Genomics, Inc. | Reversibly blocked nucleoside analogues and their use |
Non-Patent Citations (65)
Title |
---|
"Indicators", 1972, PERGAMON PRESS, OXFORD |
AHMAD ET AL., CLIN. DEV. IMMUNOL., vol. 1, 2012 |
AL QARAGHULI: "Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction", BMC BIOTECHNOLOGY, vol. 15, no. 1, 2015, pages 1, XP021230902, DOI: 10.1186/s12896-015-0217-x |
BENTLEY ET AL., NATURE, vol. 456, no. 7218, 2008, pages 53 - 59 |
BERLMAN: "Handbook of Fluorescence Spectra of Aromatic Molecules", 1971, ACADEMIC PRESS |
BEYZAVI ET AL., ANNALS CLIN BIOCHEM, vol. 24, 1987, pages 145 - 152 |
BRITTA ET AL.: "Generation of hapten-specific recombinant antibodies: Antibody phage display technology: A review", VET MED, vol. 50, 2005, pages 231 - 52 |
CHARLTON ET AL.: "Isolation of anti-hapten specific antibody fragments from combinatorial libraries", METHODS MOL BIOL, vol. 178, 2002, pages 159 - 71 |
CHEN, C.: "DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present", FRONTIERS IN MICROBIOLOGY, vol. 5, 2014, XP055174849, DOI: 10.3389/fmicb.2014.00305 |
COLE ET AL., METHODS ENZYMOL, vol. 121, 1986, pages 140 - 67 |
CONSTANS, A, THE SCIENTIST, vol. 17, no. 13, 2003, pages 36 |
CRAWFORD ET AL., BRIEF FUNCT. GENOMIC PROTEOMIC, vol. 2, 2003, pages 72 - 79 |
DESAI ET AL., ANAL BIOCHEM, vol. 294, 2001, pages 132 - 140 |
ENDO ET AL., BIOTECHNOL. ADV., vol. 21, 2003, pages 695 - 713 |
FEHLMANN ET AL., CLIN. EPIGENETICS, vol. 8, 2016, pages 123 |
GAIT: "Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS |
GENOME ANALYSIS: A LABORATORY MANUAL SERIES, vol. I-IV |
GRIFFITHS: "Colour and Constitution of Organic Molecules", 1976, ACADEMIC PRESS |
GRIMM ET AL., PROG. MOL. BIOL. TRANSL. SCI., vol. 113, 2013, pages 1 - 34 |
GUO ET AL., PROC. NATL ACAD. SCI. USA, vol. 105, 2008, pages 9145 - 9150 |
HARLOWLANE, ANTIBODIES: A LABORATORY MANUAL, 1988 |
HARLOWLANE, USING ANTIBODIES: A LABORATORY MANUAL, 1999 |
HAUGLAND: "Handbook of Fluorescent Probes and Research Chemicals", 2005, article "Molecular Probes, Eugene" |
HONGTAO ET AL.: "Molecular Modeling Application on Hapten Epitope Prediction: An Enantioselective Immunoassay for Ofloxacin Optical Isomers", J. AGRIC. FOOD CHEM., vol. 62, no. 31, 2014, pages 7804 - 7812 |
HUANG ET AL.: "A reference human genome dataset of the BGISEQ-500 sequencer", GIGASCIENCE, vol. 6, 2017, pages 1 - 9 |
HUSE ET AL., SCIENCE, vol. 246, 1989, pages 1275 |
HUSTON ET AL., METHODS ENZYMOL, vol. 203, 1991, pages 46 - 88 |
HUTTER ET AL., NUCLEOSIDES NUCLEOTIDES NUCLEIC ACIDS, vol. 29, no. 11, 2010 |
HUTTER ET AL., NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS, vol. 29, 2010, pages 879 - 895 |
INOUYE ET AL., BIOSCI BIOTECHNOL BIOCHEM, vol. 75, 2011, pages 568 - 571 |
JAYASENA ET AL., CLINICAL CHEMISTRY, vol. 45, 1999, pages 1628 - 1650 |
JU ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 19635 - 19640 |
KHANNA ET AL., MARSHALL, HISTOCHEMICAL J., vol. 7, 1975, pages 299 - 303 |
KINTZINGCOCHRAN, CURR. OPIN. CHEM. BIOL., vol. 34, 2016, pages 143 - 150 |
KNAPP ET AL., CHEM. EUR. J., vol. 17, 2011, pages 2903 - 2915 |
KOBAYASHIOYAMA, ANALYST, vol. 136, 2011, pages 642 - 651 |
KOHLERMILSTEIN, NATURE, vol. 256, 1975, pages 495 - 497 |
KOZBOR ET AL., IMMUNOL. TODAY, vol. 4, 1983, pages 72 |
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 100, 2003, pages 414 - 419 |
MARGULIES ET AL., NATURE, vol. 437, 2005, pages 376 - 380 |
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554 |
METZKER, NAT REV GENET., vol. 11, 2010, pages 31 - 46 |
MOORE ET AL., DRUG DISCOVERY TODAY: TECHNOLOGIES, vol. 9, no. 1, 2012, pages e3 - e11 |
MOORECOCHRAN, METH. ENZYMOL., vol. 503, 2012, pages 223 - 51 |
NELSONCOX: "Microarrays: A Practical Approach", 2000, FREEMAN PUB. |
OUSHIKI ET AL., ANAL. CHEM., vol. 84, 2012, pages 4404 - 4410 |
OYAMA ET AL., ANAL CHEM, vol. 87, 2015, pages 12387 - 12395 |
PINHEIRO, V ET AL.: "Protein Engineering Handbook", vol. 3, 2012, article "Polymerase Engineering: From PCR and Sequencing to Synthetic Biology", pages: 279 - 302 |
RASHIDIAN ET AL., BIOCONJUGATE CHEM, vol. 24, 2013, pages 1277 - 1294 |
RENATUS ET AL., CHEM REV., vol. 110, no. 5, 2010, pages 2579 - 2619 |
REVERDATTO ET AL., CURR. TOP. MED. CHEM., vol. 15, 2015, pages 1082 - 1101 |
RONAGHI ET AL., ANAL BIOCHEM, vol. 242, 1996, pages 84 - 89 |
RONAGHI ET AL., ANAL. BIOCHEM., vol. 242, 1996, pages 84 - 89 |
RONAGHI ET AL., SCIENCE, vol. 281, 1998, pages 363 - 365 |
RONAGHI, GENOME RES, vol. 11, 2001, pages 3 - 11 |
SCHEIT: "Nucleotide Analogs", 1980, JOHN WILEY & SON |
SHENDURE ET AL.: "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays", SCIENCE, vol. 327, 2010, pages 1728 - 1739 |
SITARAMAN ET AL., METHODS MOL. BIOL., vol. 498, 2009, pages 229 - 44 |
SKERRAPLUCKTHUN, SCIENCE, vol. 242, 1988, pages 1038 - 1041 |
SPIRIN, TRENDS BIOTECHNOL, vol. 22, 2004, pages 538 - 45 |
STRYER, L: "Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual", 1995, COLD SPRING HARBOR LABORATORY PRESS |
UHLMAN ET AL., CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584 |
WANG ET AL., ANAL CHIM ACTA, vol. 435, 2001, pages 255 - 263 |
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546 |
WHITE ET AL.: "Fluorescence Analysis: A Practical Approach", 1970, MARCEL DEKKER |
Cited By (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10851410B2 (en) | 2017-01-04 | 2020-12-01 | Mgi Tech Co., Ltd. | Nucleic acid sequencing using affinity reagents |
US11788138B2 (en) | 2017-01-04 | 2023-10-17 | Mgi Tech Co., Ltd. | Nucleic acid sequencing using affinity reagents |
JP7332235B2 (en) | 2018-11-07 | 2023-08-23 | イージーアイ テック (シェン チェン) カンパニー, リミテッド | Methods of sequencing polynucleotides |
CN112840035A (en) * | 2018-11-07 | 2021-05-25 | 深圳华大智造极创科技有限公司 | Method for sequencing polynucleotides |
EP3878968A4 (en) * | 2018-11-07 | 2022-08-17 | EGI Tech (Shen Zhen) Co., Limited | Method for sequencing polynucleotides |
JP2022513574A (en) * | 2018-11-07 | 2022-02-09 | イージーアイ テック (シェン チェン) カンパニー, リミテッド | How to sequence a polynucleotide |
CN112840035B (en) * | 2018-11-07 | 2024-01-30 | 青岛华大智造科技有限责任公司 | Method for sequencing polynucleotides |
CN113272448A (en) * | 2018-11-09 | 2021-08-17 | 深圳华大智造科技股份有限公司 | Massively parallel sequencing Using unlabeled nucleotides |
EP3877548A4 (en) * | 2018-11-09 | 2022-10-05 | MGI Tech Co., Ltd. | Massively parallel sequencing using unlabeled nucleotides |
WO2020097607A1 (en) * | 2018-11-09 | 2020-05-14 | Mgi Tech Co., Ltd. | Massively parallel sequencing using unlabeled nucleotides |
JP2022511546A (en) * | 2018-12-05 | 2022-01-31 | 深▲せん▼華大智造極創科技有限公司 | Rolling circle amplification method, sequencing library preparation method and prepared DNA nanospheres |
JP7393429B2 (en) | 2018-12-05 | 2023-12-06 | 深▲せん▼華大智造極創科技有限公司 | Rolling circle amplification method, sequencing library preparation method and prepared DNA nanospheres |
EP3702474A1 (en) * | 2019-02-26 | 2020-09-02 | QIAGEN GmbH | Sequencing method and kit |
JP7500727B2 (en) | 2019-12-18 | 2024-06-17 | エフ. ホフマン-ラ ロシュ アーゲー | Sequencing-by-synthesis method using sequential labeling schemes |
JP2023506891A (en) * | 2019-12-18 | 2023-02-20 | エフ. ホフマン-ラ ロシュ エージー. | Sequencing-by-synthesis methods using sequential labeling schemes |
US11981964B2 (en) | 2020-07-28 | 2024-05-14 | Illumina Cambridge Limited | Substituted coumarin dyes and uses as fluorescent labels |
WO2022023353A1 (en) | 2020-07-28 | 2022-02-03 | Illumina Cambridge Limited | Substituted coumarin dyes and uses as fluorescent labels |
WO2022079005A1 (en) * | 2020-10-12 | 2022-04-21 | Cambridge Display Technology Limited | Method comprising light emitting marker |
WO2022078319A1 (en) * | 2020-10-12 | 2022-04-21 | Mgi Tech Co., Ltd. | Protein detection and tracking using nucleoside tags |
WO2022083686A1 (en) | 2020-10-21 | 2022-04-28 | 深圳华大生命科学研究院 | Modified nucleoside or nucleotide |
WO2022129437A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Long stokes shift chromenoquinoline dyes and uses in sequencing applications |
WO2022129930A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Alkylpyridinium coumarin dyes and uses in sequencing applications |
WO2022129439A1 (en) | 2020-12-17 | 2022-06-23 | Illumina Cambridge Limited | Methods, systems and compositions for nucleic acid sequencing |
WO2022136402A1 (en) * | 2020-12-22 | 2022-06-30 | Illumina Cambridge Limited | Methods and compositions for nucleic acid sequencing |
WO2022206922A1 (en) | 2021-04-01 | 2022-10-06 | 深圳华大生命科学研究院 | Nucleotide analogue for sequencing |
WO2022233795A1 (en) | 2021-05-05 | 2022-11-10 | Illumina Cambridge Limited | Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing |
US12043637B2 (en) | 2021-05-05 | 2024-07-23 | Illumina Cambridge Limited | Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing |
WO2023114896A1 (en) | 2021-12-16 | 2023-06-22 | Illumina Cambridge Limited | Methods for metal directed cleavage of surface-bound polynucleotides |
WO2023122499A1 (en) | 2021-12-20 | 2023-06-29 | Illumina Cambridge Limited | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides |
WO2023122491A1 (en) | 2021-12-20 | 2023-06-29 | Illumina Cambridge Limited | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides |
WO2023186819A1 (en) | 2022-03-29 | 2023-10-05 | Illumina Cambridge Limited | Chromenoquinoline dyes and uses in sequencing |
WO2023192163A1 (en) | 2022-03-29 | 2023-10-05 | Illumina Cambridge Limited | Systems and methods of sequencing polynucleotides |
WO2023186872A1 (en) | 2022-03-30 | 2023-10-05 | Illumina Cambridge Limited | Methods for chemical cleavage of surface-bound polynucleotides |
WO2023186982A1 (en) | 2022-03-31 | 2023-10-05 | Illumina, Inc. | Compositions and methods for improving sequencing signals |
WO2023192900A1 (en) | 2022-03-31 | 2023-10-05 | Illumina Singapore Pte. Ltd. | Nucleosides and nucleotides with 3' vinyl blocking group useful in sequencing by synthesis |
WO2024003087A1 (en) | 2022-06-28 | 2024-01-04 | Illumina, Inc. | Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing |
WO2024068889A2 (en) | 2022-09-30 | 2024-04-04 | Illumina, Inc. | Compositions and methods for reducing photo damage during sequencing |
WO2024123866A1 (en) | 2022-12-09 | 2024-06-13 | Illumina, Inc. | Nucleosides and nucleotides with 3´ blocking groups and cleavable linkers |
WO2024137765A1 (en) | 2022-12-22 | 2024-06-27 | Illumina, Inc. | Transition-metal catalyst compositions and methods for sequencing by synthesis |
WO2024137774A1 (en) | 2022-12-22 | 2024-06-27 | Illumina, Inc. | Palladium catalyst compositions and methods for sequencing by synthesis |
WO2024145154A1 (en) | 2022-12-27 | 2024-07-04 | Illumina, Inc. | Methods of sequencing using 3´ allyl blocked nucleotides |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2018205472B2 (en) | Nucleic acid sequencing using affinity reagents | |
US20220162693A1 (en) | Massively parallel sequencing using unlabeled nucleotides | |
CN107614700A (en) | Genotype and phenotype coupling | |
JP2023551072A (en) | Multiplexed profiling of RNA and DNA modifications | |
CA3112661A1 (en) | Nucleotide analogs | |
CN101952719A (en) | Detection assay method and uses thereof | |
JP6506838B2 (en) | High-speed photocrosslinkable co-linker for in-vitro sputum and intermolecular interaction analysis, in-vitro sputum method using the linker | |
US11236342B2 (en) | Secretory immunoglobulin a (sIgA)-binding nucleic acid molecule, sIgA analysis sensor, and sIgA analysis method | |
WO2016061460A1 (en) | Enhanced biomolecule detection assays based on tyramide signal amplification and gammapna probes | |
US20210395742A1 (en) | Compositions and methods for the synthesis and identification of covalent aptamers | |
RU2699522C2 (en) | Method for enzymatic production of modified dna for producing reagents which specifically bind to hydrophobic areas of high molecular weight organic compounds | |
AU2022397404A1 (en) | Rna and dna analysis using engineered surfaces | |
WO2022078319A1 (en) | Protein detection and tracking using nucleoside tags | |
US20060188958A1 (en) | Artificial enzyme and method for producing the same | |
Wu | Synthetic Nucleic Acid Capable of Post-Polymerization Functionalization and Evolution | |
JP6194894B2 (en) | Nucleic acid linker |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18736171 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3048486 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2019536551 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112019013715 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 2018205472 Country of ref document: AU Date of ref document: 20180104 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20197022620 Country of ref document: KR Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2018736171 Country of ref document: EP Effective date: 20190805 |
|
ENP | Entry into the national phase |
Ref document number: 112019013715 Country of ref document: BR Kind code of ref document: A2 Effective date: 20190702 |