CN109134541A - Long-chain biological element marker and its preparation method and application - Google Patents
Long-chain biological element marker and its preparation method and application Download PDFInfo
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- CN109134541A CN109134541A CN201811051423.0A CN201811051423A CN109134541A CN 109134541 A CN109134541 A CN 109134541A CN 201811051423 A CN201811051423 A CN 201811051423A CN 109134541 A CN109134541 A CN 109134541A
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- 239000003550 marker Substances 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 78
- 229960002685 biotin Drugs 0.000 claims abstract description 39
- 235000020958 biotin Nutrition 0.000 claims abstract description 39
- 239000011616 biotin Substances 0.000 claims abstract description 39
- 238000012163 sequencing technique Methods 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 14
- 239000011574 phosphorus Substances 0.000 claims abstract description 14
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N pentanoic acid group Chemical group C(CCCC)(=O)O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 8
- 239000001301 oxygen Substances 0.000 claims abstract description 8
- 125000004122 cyclic group Chemical group 0.000 claims abstract description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000013067 intermediate product Substances 0.000 claims description 58
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- 108091034117 Oligonucleotide Proteins 0.000 claims description 40
- 239000000523 sample Substances 0.000 claims description 33
- 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 claims description 23
- 238000000034 method Methods 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 13
- 150000003536 tetrazoles Chemical class 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- -1 chloro-carbonic acid p-nitrophenyl phenolic ester Chemical class 0.000 claims description 11
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- ASDQMECUMYIVBG-UHFFFAOYSA-N 2-[2-(2-aminoethoxy)ethoxy]ethanol Chemical compound NCCOCCOCCO ASDQMECUMYIVBG-UHFFFAOYSA-N 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- NPEIGRBGMUJNFE-UHFFFAOYSA-N 1-aminohexan-1-ol Chemical compound CCCCCC(N)O NPEIGRBGMUJNFE-UHFFFAOYSA-N 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- ZJIFDEVVTPEXDL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) hydrogen carbonate Chemical class OC(=O)ON1C(=O)CCC1=O ZJIFDEVVTPEXDL-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000002777 nucleoside Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 2
- 125000003835 nucleoside group Chemical group 0.000 claims description 2
- ZLRAAUUPULJGTL-UHFFFAOYSA-N diaminophosphinous acid Chemical compound NP(N)O ZLRAAUUPULJGTL-UHFFFAOYSA-N 0.000 claims 1
- 239000000090 biomarker Substances 0.000 abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 84
- 108020004414 DNA Proteins 0.000 description 17
- 239000012043 crude product Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 238000012986 modification Methods 0.000 description 12
- 230000004048 modification Effects 0.000 description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 238000002390 rotary evaporation Methods 0.000 description 10
- 239000000741 silica gel Substances 0.000 description 10
- 229910002027 silica gel Inorganic materials 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 239000005289 controlled pore glass Substances 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 230000009514 concussion Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 6
- 239000012925 reference material Substances 0.000 description 5
- 235000005078 Chaenomeles speciosa Nutrition 0.000 description 4
- 240000000425 Chaenomeles speciosa Species 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- PFYXSUNOLOJMDX-UHFFFAOYSA-N bis(2,5-dioxopyrrolidin-1-yl) carbonate Chemical compound O=C1CCC(=O)N1OC(=O)ON1C(=O)CCC1=O PFYXSUNOLOJMDX-UHFFFAOYSA-N 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 2
- BSKHPKMHTQYZBB-UHFFFAOYSA-N 2-methylpyridine Chemical compound CC1=CC=CC=N1 BSKHPKMHTQYZBB-UHFFFAOYSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- WZELXJBMMZFDDU-UHFFFAOYSA-N Imidazol-2-one Chemical group O=C1N=CC=N1 WZELXJBMMZFDDU-UHFFFAOYSA-N 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical compound OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000008236 heating water Substances 0.000 description 2
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Substances [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 125000004437 phosphorous atom Chemical group 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 2
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- LELMRLNNAOPAPI-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;aminophosphonous acid Chemical compound NP(O)O.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LELMRLNNAOPAPI-UFLZEWODSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- WHLUQAYNVOGZST-UHFFFAOYSA-N tifenamil Chemical group C=1C=CC=CC=1C(C(=O)SCCN(CC)CC)C1=CC=CC=C1 WHLUQAYNVOGZST-UHFFFAOYSA-N 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
- C07F9/65618—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system, e.g. flavins or analogues
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
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- Proteomics, Peptides & Aminoacids (AREA)
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- Analytical Chemistry (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The present invention discloses long-chain biological element marker and its preparation method and application.Long-chain biological element marker of the invention includes biotin molecule, spacerarm and reactive group.Wherein, biotin molecule includes cyclic structure and pentanoic acid side chain, spacerarm has structure shown in formula (I), reactive group has structure shown in formula (II), pentanoic acid side chain in biotin molecule is connect with the NH of spacerarm first end, the oxygen of the second end of spacerarm forms O-P key in conjunction with the phosphorus of reactive group, so that spacerarm be connect with reactive group.Long-chain biological marker of the invention can be used for the capture sequencing of gene.-NH(CH2)mO- (- C=O-NH- (CH2‑CH2O‑)4)nFormula (I)
Description
Technical field
The present invention relates to the biomarkers for gene sequencing, and in particular to is used for gene sequencing, especially two generations survey
Long-chain biological element marker of sequence and its preparation method and application.
Background technique
Increasingly important role is played in the sequencing of two generations in molecular biology and clinical medicine.Wherein, target area is caught
Obtaining sequencing is the very high means of cost performance in the sequencing application of two generations, in genetic disease and tumour scientific research and clinical diagnosis
There is wide application space in field.Its principle is the complementary probe for interested genome area design specificity, will
It is hybridized with genomic DNA, is enriched with the DNA fragmentation of target genome area, then carries out high-flux sequence.
The committed step of capture enrichment is caught in genome and is felt using the single stranded DNA of biotin modification as capture reagent
Then the target area of interest utilizes the natural specificity combination energy of streptavidin (streptavidin, SA) and biotin
Power is separated in magnetic field in conjunction with the magnetic bead of SA modification.
Areas captured can be divided into solid-phase hybridization method and solution hybridization method by state when according to hybridization.Used in solid-phase hybridization method
Probe be usually all to be fixed on solid support, such as genetic chip.The DNA fragmentation on non-hybridized is first eluted after hybridization,
The DNA hybridized with probe is eluted again.Now more popular is solution hybridization.It is with solid-phase hybridization maximum difference
Solution hybridization makes target DNA fragments and the probe direct cross with biotin labeling in the solution.It is public using the technology at present
Department includes mainly foreign biomolecule scientific & technical corporation.Such as Agilent, Roche, match are silent winged.
Existing biotin modification can pass through dUTP (the Deoxyuridine Triphosphate of addition biotin modification
Deoxyuridine triphosphate) the method random doping that is replicated is in DNA chain.The affinity combined between biotin and avidin
High, high specificity.There are two cyclic structures for biotin molecule, and wherein imidazolone ring is the main portions in conjunction with Avidin；Separately
There is a pentanoic acid side chain on the C2 of an outer thiphene ring, certain space length can be provided, but this side chain lengths are simultaneously insufficient
It is enough.Therefore, biotin generally passes through organic long-chain shape molecule with DNA and connects, to adjust its distance.What is generallyd use has hexane
Chain, triethylene glycol (TEG) chain or chain of other length etc., for example, the Biotin-16-dUTP (CAS mixed when DNA replication dna
Number 136632-31-0) using the organic molecule chain of 16 atomic lengths.However, these organic long-chains can not be complete
Meet the needs of current, the especially demand in terms of the sequencing of two generations.
Summary of the invention
To solve at least partly technical problem in the prior art, the present invention have studied biotin molecule and oligonucleotides it
Between influence of the organic long-chain structure for the probe capture in the sequencing of two generations, be found to have the long-chain biological element point of specific structure
Son is sequenced particularly suitable for two generations, and within the scope of certain chain lengths, capture rate is stepped up.It is based at least partially on above-mentioned
Content completes the present invention.Specifically, the present invention includes the following contents.
The first aspect of the present invention provides a kind of long-chain biological element marker comprising biotin molecule, spacerarm and anti-
Answer group, wherein the biotin molecule includes cyclic structure and pentanoic acid side chain, and the spacerarm has to be tied shown in formula (I)
Structure, the reactive group have structure shown in formula (II), the first end of pentanoic acid side chain and spacerarm in the biotin molecule
The NH connection at end, the oxygen of the second end of the spacerarm form O-P key in conjunction with the phosphorus of the reactive group, thus will be described
Spacerarm is connect with the reactive group,
-NH(CH2)mO- (- C=O-NH-CH2-CH2O)nFormula (I)
M and n separately indicates the integer of 1-10 in formula (I),
In certain embodiments, the biotin molecule in long-chain biological element marker of the invention is by DMT radical protection.
In certain embodiments, the m in long-chain biological element marker of the invention in formula (I) is the integer of 4-6, and n
For the integer of 1-3.
The second aspect of the present invention provides the preparation method of long-chain biological element marker described in first aspect present invention,
Comprising:
(1) in pyridine solution, biotin and DMT-Cl is made to react to obtain intermediate product A；
(2) in anhydrous dimethyl formamide, intermediate product A is made to react to obtain intermediate product B with amino-hexanol；
(3) in anhydrous acetonitrile, with chloro-carbonic acid p-nitrophenyl phenolic ester activate intermediate product B, later with amino-PEG3- alcohol
Reaction obtains intermediate product C1, replaces intermediate product B to repeat the step again n-1 times with intermediate product C1, obtains intermediate production
Object Cn, wherein n is the integer of 1-10；
(4) in anhydrous acetonitrile, intermediate product Cn is made to react to obtain long-chain biological element with phosphorus reagent under tetrazole effect
Marker.
In certain embodiments, use in the step of method of the invention (2) two succinimidyl carbonates as
Catalyst.
In certain embodiments, the phosphorus reagent in the step of method of the invention (4) is 2- cyanoethyl-N, N, N ' N '-
Tetraisopropylphosph-ro phosphoryl diamine.
The third aspect of the present invention provides long-chain biological element marker described in first aspect present invention and is preparing gene survey
Purposes in sequence/detection probe.
The fourth aspect of the present invention provides a kind of oligonucleotides comprising long-chain biological described in first aspect present invention
Plain marker and oligonucleotide sequence, wherein the long-chain biological element marker is bound to the hydroxyl at the end of oligonucleotide sequence 5 '.
The fifth aspect of the present invention provides a kind of preparation method of oligonucleotides comprising:
The step of (1 ') is by multiple nucleotide sequence synthetic oligonucleotides；(2 ') long-chain biological element marker combines step
Suddenly comprising use tetrazole admixture activation long-chain biological element marker, the oligonucleotides sequence for obtaining it with step (1)
The hydroxyl of column the last one base of the end 5' is reacted；Then it is aoxidized and DMT is gone to protect.
The sixth aspect of the present invention provides long-chain biological element marker described in first aspect present invention or the present invention the 4th
Purposes of the oligonucleotides described in aspect in two generations were sequenced.
Long-chain biological element marker of the invention can be used for the capture sequencing of specific gene.By sequence verification, capture effect
Rate can achieve 80% or more, can be efficiently used for the target area gene sequencing customized.
Detailed description of the invention
The molecular structure of the existing Biotin-16-dUTP of Fig. 1 (CAS number 136632-31-0).
The peak figure for the molecule 1-3 product that Fig. 2 present invention obtains.Its for HPLC after purification, according to UV absorption at 260nm
Light absorption value, figure is done to the retention time of elution.
The peak figure of Fig. 3 reference material, after purification for HPLC, according to light absorption value of the UV absorption at 260nm, to elution
Retention time does figure.
The figure that the product and two figure of reference material that Fig. 4 present invention obtains obtain after merging.
The Mass Spectrometric Identification figure of the product of Fig. 5 long-chain biological element modification.
Fig. 6 difference biotin modification probe hybrid capture efficiency comparative.Three columns form one group in figure.Left side in every group of column
Column indicate the ratio that is captured to of target area.Intermediate column indicates to compare the base ratio for arriving target area.The column on right side
It indicates to compare the read ratio for arriving target area.
Specific embodiment
The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair
It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it
Between each median.Median and any other statement value in any statement value or stated ranges or in the range
Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent
Ground includes or excludes in range.
Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention
The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention
Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification
There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any
When document conflicts, it is subject to the content of this specification.Unless otherwise stated, " % " is the percentage based on weight.
[long-chain biological element marker]
The first aspect of the present invention provides a kind of long-chain biological element marker.It is the biotin molecule with long side chain
(being sometimes referred to as long-chain biological element molecule herein), due to can be used as the molecular marked compound in the fields such as gene sequencing, herein
Referred to as long-chain biological element marker.Compared with general biotinylated derivative, long-chain biological element marker of the invention at least has
Longer side chain and higher activity.Preferably, the side chain in long-chain biological element marker of the invention does not include branched structure,
Especially carbon branched structure does not include any cyclic structure, such as phenyl ring or cyclic alkyl structure yet.In addition, long-chain of the invention
The side chain of biotinylated derivative does not also include carbon-carbon double bond.Present invention discover that branched structure, cyclic structure and carbon-carbon double bond etc.
Although side chain can be made longer, these structures influence side chain flexibility, and oligonucleotides is influenced after being bound to oligonucleotides
It is complementary the combination of sequence, therefore, above-mentioned group is preferably not included in spacer group.
Long-chain biological marker of the invention includes biotin molecule, spacerarm and reactive group.Wherein, biotin molecule
Including cyclic structure and pentanoic acid side chain, spacerarm has structure shown in formula (I), and reactive group has to be tied shown in formula (II)
Structure.As shown in formula (I), spacerarm has the first extended segment and the second extended segment, and an end of the first extended segment is (that is, first
End) there is NH group.Second extended segment is repeated segments.One end (that is, second end) is oxygen.In biotin molecule
Pentanoic acid side chain is connect with the NH of spacerarm first end.The oxygen of the second end of spacerarm forms O- in conjunction with the phosphorus of reactive group
P key, so that spacerarm be connect with reactive group.It should be noted that the connection of valeric acid and spacerarm first end passes through carboxylic
Base reacts to obtain with amido.Therefore, the pentanoic acid side chain in biotin molecule connect with the NH of spacerarm first end be by-
(C=O)-NH- is realized.
-NH(CH2)mO- (- C=O-NH-CH2-CH2O)nFormula (I)
In formula (I) of the invention, m and n separately indicate the integer of 1-10.In order to improve capture rate, it is preferable that
M is the integer of 3-10, and more preferable m is the integer of 4-8.For example, m can be 5,6 or 7.In addition, n is preferably the integer of 1-5, it is more excellent
It is selected as the integer of 1-4.For example, n can be 1,2,3 or 4 etc..If m or n is greater than above range, on the one hand make to synthesize difficulty increase,
On the other hand further increase with chain length, in conjunction with oligonucleotides after, it is unobvious for the capture effect of oligonucleotides
It improves.Therefore, m of the invention and n is preferably within the above range.
In formula (II) of the invention, reactive group includes cyanoethyl and two isopropyls.Reactive group with the structure
It is particularly conducive to carry out necleophilic reaction with the hydroxyl in nucleic acid, by the phosphorus atoms in the hydroxyl radicals attack formula (II) in nucleic acid, from
And guaranteeing long-chain biological element marker has higher reactivity.
In certain embodiments, the biotin molecule of long-chain biological element marker of the invention is by DMT radical protection.
Preferably, DMT group is in conjunction with the N in the imidazolone ring of biotin.Preferably, DMT group is for protecting biotin molecule
That more active NH in two NH.
[preparation method of long-chain biological element marker]
The second aspect of the present invention provides the preparation method of long-chain biological element marker comprising at least following four steps
Suddenly.
Step (1)
Step (1) of the invention be carry out synthetic reaction before, to biotin molecule carry out DMT protection the step of.Specifically,
It is included in pyridine solution, biotin and DMT-Cl is made to react to obtain intermediate product A.More specifically, make raw material biotin and
DMT-Cl is dissolved in pyridine solution according to the mole of 1:2-1:5,50-90 DEG C (such as 80 DEG C) heating water bath 1-8 hours, it is excellent
2-6 hours, such as 4 hours are selected, crude product is obtained.Then water is added in crude product and methylene chloride, concussion shakes up, isolate two
It is dry that anhydrous sodium sulfate is added in chloromethanes layer.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains
Between product A.
Step (2)
Step (2) of the invention is the step of obtaining the first extended segment.That is, in pentanoic acid side chain end addition amino-hexanol
Step.Known method can be used to carry out in the present invention.Preferably, step of the invention (2) is included in anhydrous dimethyl formamide
In, make intermediate product A react to obtain intermediate product B with amino-hexanol.It is highly preferred that step (2) includes being dissolved in intermediate product A
In anhydrous DMF (dimethylformamide), in two succinimidyl carbonate DSC (disuccinimidyl carbonate) etc.
It is reacted 1 hour under catalyst action with 1:1 mole amino-hexanol, is condensed to yield crude product.Water and methylene chloride, shake is added
It swings and shakes up, isolate dichloromethane layer, it is dry that anhydrous sodium sulfate is added.After dichloromethane layer rotary evaporation, mixture is in silica gel
It is separated on column, obtains intermediate product B.
Step (3)
Step (3) of the invention is the step of obtaining the second extended segment.That is, addition-C=O-NH- (CH in the side chain2-
CH2O)4The step of constitutional repeating unit.- C=O-NH- (CH in side chain2-CH2O)4The repeat number n of constitutional repeating unit is unlimited
It is fixed.The meaning of n is referring to the explanation for being directed to formula (I).
Step (3) of the invention may include activating intermediate product B with chloro-carbonic acid p-nitrophenyl phenolic ester in anhydrous acetonitrile,
It reacts to obtain intermediate product C1 with amino-PEG3- alcohol later.Intermediate product B is replaced to repeat the step again with intermediate product C1
It is n-1 times rapid, intermediate product Cn is obtained, wherein the meaning of n is identical in the meaning with formula (I) of n.
In certain embodiments, the n in step (3) is 1, then step (3) is only included in anhydrous acetonitrile, uses chloro-carbonic acid
P-nitrophenyl phenolic ester activates intermediate product B, reacts to obtain intermediate product C1 with amino-PEG3- alcohol later, without carrying out below
Repetition step.Only contain-C=O-NH- (a CH in intermediate product C1 at this time2-CH2O)4Structural unit.
In certain embodiments, the n in step (3) is 2, then step (3) is included in anhydrous acetonitrile, with chloro-carbonic acid pair
Nitrobenzene phenolic ester activates intermediate product B, reacts to obtain intermediate product C1 with amino-PEG3- alcohol later.Hereafter, with intermediate product
C1 replaces intermediate product B to repeat the step 1 time again, obtains intermediate product C2.It include two-C=O-NH- in intermediate product C2
(CH2-CH2O)4Structural unit.
In certain embodiments, the n in step (3) is 3, then step (3) is included in anhydrous acetonitrile, with chloro-carbonic acid pair
Nitrobenzene phenolic ester activates intermediate product B, reacts to obtain intermediate product C1 with amino-PEG3- alcohol later.Hereafter, with intermediate product
C1 replaces intermediate product B to repeat the step 1 time, obtains intermediate product C2.Then with intermediate product C2 instead of intermediate product C1 weight
Multiple step 1 time, obtains intermediate product C3.It include three-C=O-NH- (CH in intermediate product C32-CH2O)4Structural unit.
The rest may be inferred, and n-C=O-NH- (CH are readily understood in those skilled in the art2-CH2O)4The side chain of structural unit.
Step (4)
Step (4) of the invention is the step of the side chain terminal of last intermediate product Cn adds reactive group.It is wrapped
It includes in anhydrous acetonitrile, intermediate product Cn is made to react to obtain long-chain biological element marker with phosphorus reagent under tetrazole effect.Tool
Body, including intermediate product Cn is dissolved in anhydrous acetonitrile, under tetrazole catalysis and 1:1 mole phosphorus reagent reacts 10 points
Clock obtains crude product.Water and methylene chloride is added, shakes up, isolates dichloromethane layer, it is dry that anhydrous sodium sulfate is added.Dichloro
After methane layer rotary evaporation, mixture separates on a silica gel column, obtains long-chain biological element molecule of the invention.In step (4)
Phosphorus reagent is preferably 2- cyanoethyl-N, N, N ' N '-tetraisopropylphosph-ro phosphoryl diamine.
[the first purposes]
The third aspect of the present invention be first aspect present invention described in long-chain biological element marker preparation gene sequencing/
Purposes in detection probe.It is referred to as the first purposes herein.Long-chain biological element marker of the invention can be with oligonucleotides sequence
Column combine and obtain oligonucleotides as described herein, can be used as molecular marked compound.Preferably, which is gene sequencing
Or probe when detection.It is highly preferred that the capture of long-chain biological element marker of the invention for specific gene is sequenced, especially
It is efficiently used for the target area gene sequencing customized.
[oligonucleotides]
The fourth aspect of the present invention provides oligonucleotides.Oligonucleotides of the invention includes described in first aspect present invention
Long-chain biological element marker and oligonucleotide sequence.Wherein long-chain biological element marker is bound to the end of oligonucleotide sequence 5 '
Hydroxyl.For long-chain biological element marker in the hydroxy combining held with oligonucleotide sequence 5 ', the hydroxyl radicals attack long-chain by 5 ' ends is raw
P atom in the reactive group of object element marker makes reactive group be detached from long-chain biological element marker, while in conjunction with P atom
Oxygen atom with 5 ' end hydroxyl react to form oligonucleotides of the invention.
The sequence length of oligonucleotide sequence of the invention is generally 50-150bp, preferably 55-100bp, more preferable 60-
80bp.The oligonucleotide sequence of long-chain biological element marker of the invention especially suitable for above-mentioned length range.If few nucleosides
Acid sequence length is too short, although then biotinylated derivative and oligonucleotide molecules have enough spacing distances, due to life
Object element marker molecules are close with oligonucleotide sequence bulk of molecule, influence oligonucleotide sequence leaning on to target gene sequence
Closely, it and then influences to specifically bind with target gene sequence.On the other hand, it if oligonucleotide length is too long, detects sensitive
Degree reduces.
[preparation method of oligonucleotides]
The fifth aspect of the present invention provides the preparation method of oligonucleotides, at least includes the following steps.
Step (1 ')
The step of step (1 ') of the invention is by multiple nucleotide sequence synthetic oligonucleotides.The step of synthetic oligonucleotide
Suddenly usable methods known in the art carry out.Specific method includes solid-phase synthesis etc..In exemplary synthetic procedure, packet
It includes:
The first step, trichloroacetic acid (TCA) and is connected to solid phase carrier (preferably, controlled pore glass controlled- in advance
Pore glass, abbreviation CPG) on first nucleotide reaction (preferably, active group be in by guard mode), slough
The blocking group DMT of nucleotide 5'- hydroxyl obtains free 5'- hydroxyl；
Second step, tetrazole admixture activation base phosphoramidite monomer (Nucleoside Phosphoramidite), with
5'- hydroxyl above CPG is reacted；
Third step, band cap (capping) react, and may have only a few 5'- hydroxyl not participate in reaction in condensation reaction (few
In 2%), continue to react thereafter with acetic anhydride and the termination of 1- methylimidazole；
Phosphorous acyl-oxygen is turned to stable phosphotriester with iodo- oxidation of methylpyridine agent by the 4th step；
Repeat above-mentioned steps, until having synthesized required oligonucleotide sequence (DNA sequence dna).It can be in synthesis process
The color for observing TCA processing stage determines combined coefficient.
Step (2 ')
Step (2 ') of the invention is long-chain biological element marker combination step comprising long using tetrazole admixture activation
The hydroxyl of chain biotinylated derivative, the 5 ' base of oligonucleotide sequence for obtaining it with step (1) is reacted；Then into
Row aoxidizes and DMT is gone to protect.
[the second purposes]
The sixth aspect of the present invention provides long-chain biological element marker described in first aspect present invention or the present invention the 4th
Purposes of the oligonucleotides described in aspect in two generations were sequenced.It is referred to as the second purposes herein.Second purposes of the invention includes using
Make probe when gene sequencing or detection.Preferably, long-chain biological element marker of the invention is surveyed for the capture of specific gene
Sequence is especially efficiently used for the target area gene sequencing customized.
Other than substance itself is different, the first purposes and the second purposes of the invention can be identical.
Embodiment 1
The synthesis of biotin molecule:
1. the synthesis of molecule 1
The synthetic line of molecule 1 is as follows:
Step 1: raw material biotin and DMT-Cl are dissolved in pyridine solution according to 1:3 mole, 80 DEG C of heating water baths 4
Hour, obtained crude product.Water is added into gained crude product and methylene chloride, concussion shake up, isolates dichloromethane layer, adds
It is dry to enter anhydrous sodium sulfate.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains intermediate product A.
Step 2: intermediate product A is dissolved in anhydrous dimethyl formamide, under DSC catalysis and 1:1 mole amino
Hexanol reacts 1 hour, is condensed to yield crude product.Water is added to crude product and methylene chloride, concussion shake up, isolates methylene chloride
It is dry that anhydrous sodium sulfate is added in layer.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains intermediate product
B。
Step 3: intermediate product B is dissolved in anhydrous acetonitrile, under tetrazole catalysis and 1:1 mole phosphorus reagent reacts 10
Minute, obtain crude product.Water is added into crude product and methylene chloride, concussion shake up, isolates dichloromethane layer, is added anhydrous
Sodium sulphate is dry.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains molecule 1.By molecule in the present invention
1 is used as comparative example.
2. molecule 2 synthesizes
The synthetic route of molecule 2 is as follows:
Step 1: intermediate product B is dissolved in anhydrous acetonitrile, with after the activation of chloro-carbonic acid p-nitrophenyl phenolic ester and 1:1 moles
It measures amino-PEG3- alcohol to react 10 minutes, obtains crude product.Water is added and methylene chloride, concussion shake up, isolates methylene chloride
It is dry that anhydrous sodium sulfate is added in layer.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains intermediate product
C1。
Step 2: intermediate product C1 is dissolved in anhydrous acetonitrile, reacted under tetrazole catalysis with 1:1 mole phosphorus reagent
10 minutes, obtain crude product.Water is added and methylene chloride, concussion shake up, isolates dichloromethane layer, it is dry that anhydrous sodium sulfate is added
It is dry.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains molecule 2.
3. the synthesis of molecule 3
The synthetic line of molecule 3 is as follows:
Step 1: intermediate product C1 is dissolved in anhydrous acetonitrile, with after the activation of chloro-carbonic acid p-nitrophenyl phenolic ester and 1:1 moles
It measures amino-PEG3- alcohol to react 10 minutes, obtains crude product.Water is added and methylene chloride, concussion shake up, isolates methylene chloride
It is dry that anhydrous sodium sulfate is added in layer.After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains intermediate product
C2；
Step 2: intermediate product C2 is dissolved in anhydrous acetonitrile, reacted under tetrazole catalysis with 1:1 mole phosphorus reagent
10 minutes, obtain crude product.Water and methylene chloride is added, shakes up, isolates dichloromethane layer, it is dry that anhydrous sodium sulfate is added.
After dichloromethane layer rotary evaporation, mixture separates on a silica gel column, obtains molecule 3.
Embodiment 2
One, the DNA synthesis of long-chain biological element molecule:
The first step, trichloroacetic acid (TCA) and first nucleotide being connected on controlled pore glass (CPG) in advance are anti-
It answers, wherein active group is in by guard mode, is sloughed the blocking group DMT of nucleotide 5'- hydroxyl, is obtained free 5'- hydroxyl
Base；
Second step, tetrazole admixture activation base phosphoramidite monomer, is reacted with the 5'- hydroxyl above CPG；
Third step, band cap react, and may have only a few 5'- hydroxyl not participate in reaction (less than 2%) in condensation reaction, use
Acetic anhydride and the termination of 1- methylimidazole continue to react thereafter；
Phosphorous acyl-oxygen is turned to stable phosphotriester with iodo- oxidation of methylpyridine agent by the 4th step；
Repeat above-mentioned step, until the oligonucleotide sequence (Oligo DNA) of length needed for synthesizing.Synthesis process
In can observe TCA processing stage color determine combined coefficient.Last tetrazole admixture activation biotin phosphoramidite monomer,
It is reacted with the 5'- hydroxyl of the last one base of DNA above CPG；Then aoxidized and gone DMT to protect step.
Two, the DNA of long-chain biological element molecule isolating and purifying and identifies:
After synthesis, Oligo DNA molecular is cut from CPG with ammonium hydroxide, then carry out HPLC purifying.HPLC purifies institute
The product obtained carries out Mass Spectrometric Identification, to differentiate whether long-chain biological element successfully connects on the 5 ' ends of DNA.
Embodiment 3
One, the DNA of long-chain biological element molecular modification is for capturing experiment
Experimental design: different length biotin modification probe design synthesis people target area (BoKe oncogene is used
Panel) capture probe；People's standard gene group DNA library is constructed, cooperates 9 kinds of different library labels, referring to table 1.It uses respectively
The probe that molecule 1,2,3 is modified carries out captive test.
1, table is tested label used
| Label | Sequence |
| Library label 1 | CGATGT |
| Library label 2 | TGACCA |
| Library label 3 | ACAGTG |
| Library label 4 | GCCAAT |
| Library label 5 | CAGATC |
| Library label 6 | CTTGTA |
| Library label 7 | ATCACG |
| Library label 8 | TTAGGC |
| Library label 9 | ACTTGA |
(1) BoKe oncogene Panel probe synthesizes
Probe design is carried out for BoKe oncogene Panel, the target area Panel overall length is 1190775bp, and probe is total
It number 17448, is respectively synthesized molecule 1, molecule 2 and molecule 3 and modifies probe.
(2) library construction
People standard gene group DNA cooperation Bio Scientific company NEXT flex Rapid DNA-Seq Kit builds library
Kit is carried out according to standard operating procedure, and library sorts segment 350bp or so.
(3) target area captures
Hybrid capture is carried out according to Boke hybrid capture experiment flow, molecule 1, molecule 2 and molecule 3 modify probe and respectively do 3
A parallel test, as shown in table 2.
2 hybrid capture test prod of table
| Bank number | Probe type |
| Boke_lib001_S01 | Molecule 1 modifies probe |
| Boke_lib001_S02 | Molecule 1 modifies probe |
| Boke_lib001_S03 | Molecule 1 modifies probe |
| Boke_lib001_S04 | Molecule 2 modifies probe |
| Boke_lib001_S05 | Molecule 2 modifies probe |
| Boke_lib001_S06 | Molecule 2 modifies probe |
| Boke_lib001_S07 | Molecule 3 modifies probe |
| Boke_lib001_S08 | Molecule 3 modifies probe |
| Boke_lib001_S09 | Molecule 3 modifies probe |
(4) machine is sequenced on
Library is sequenced using HiSeq X Ten afterwards through Quality Control (library fragments < 500bp, concentration 3-5nM) after capture,
PE150 mode, the estimated sequencing data amount 2G base of each sample.
(5) data
After purification, the peak figure of product is as shown in Figure 2 by HPLC.According to light absorption value of the UV absorption at 260nm, product is washed
De- retention time does figure.37 minutes maximum absorption bands gone out are collected, the length for obtaining the modification of long-chain biological element is 60 bases
Single-stranded DNA product.
After purification, the peak figure of reference material is as shown in Figure 3 by HPLC.According to light absorption value of the UV absorption at 260nm, to control
The retention time of object elution does figure.In the maximum absorption band that 30-31 minutes go out, the length for being free from biotin modification is 60 bases
Single-stranded DNA product.
After purification HPLC, the two above peak figure of product and reference material merges, and obtains comparison figure as shown in Figure 4.It can
, it is evident that the product containing long-chain biological element and the reference material without long-chain biological element, can be had on a column
The separation of effect.
Two, Mass Spectrometric Identification
After purification, the peak figure of the product of long-chain biological element modification collects 37 minutes maximum absorption bands gone out to HPLC, carries out matter
Spectrum identification.
The oligonucleotide sequence of product is (5 ' to 3 ') as follows:
TCTCGGCTCCTCATGACCGCCATGTTCTTCTCCTCACTGAGCTGTGCGTAGCGCATGGCT
Molecular weight theoretical value: 19128, measured value: 19130, mass spectrogram is as shown in Figure 5.Pass through HPLC separating-purifying, matter
Show to synthesize successfully after spectrum identification.
Three, the probe of different molecular modification is used to capture the sequencing result of experiment
It is for statistical analysis to sequencing data using BWA, Samtools, Picard and perl script program, it the results are shown in Table 3
And Fig. 6.It will be seen from figure 6 that the probe that probe and molecule 1 that molecule 2 and molecule 3 are modified are modified is being compared to target area
Base ratio and/or compare to target area read (reads) ratio two indices on it is significantly superior.Higher comparison is arrived
The base ratio of target area and read (reads) ratio compared to target area imply and less sequencing number can be used
Reach effective covering to target area according to amount, and then sequencing cost can be reduced, shortens the data-analysis time.
The different hybrid capture library sequencing datas of table 3 analyze result
Experiment is sequenced in capture for specific gene, shows that capture rate is significantly improved than common short chain biotin, catches
Obtaining efficiency can achieve 80% or more, can be effective for the target area gene sequencing of customization.
Without departing substantially from the scope or spirit of the invention, the specific embodiment of description of the invention can be done more
Kind improvements and changes, this will be apparent to those skilled in the art.Other realities obtained by specification of the invention
Applying mode for technical personnel is apparent obtain.Present specification and embodiment are merely exemplary.
Claims (10)
1. a kind of long-chain biological element marker comprising biotin molecule, spacerarm and reactive group, wherein the biotin
Molecule includes cyclic structure and pentanoic acid side chain, and the spacerarm has structure shown in formula (I), and the reactive group has formula
(II) structure shown in, the pentanoic acid side chain in the biotin molecule are connect with the NH of spacerarm first end, the spacerarm
The oxygen of second end O-P key is formed in conjunction with the phosphorus of the reactive group, thus by the spacerarm and the reactive group
Connection,
-NH(CH2)mO- (- C=O-NH- (CH2-CH2O-)4)nFormula (I)
M and n separately indicates the integer of 1-10 in formula (I),
2. long-chain biological element marker according to claim 1, wherein the biotin molecule is by DMT radical protection.
3. long-chain biological element marker according to claim 2, wherein the integer that the m in the formula (I) is 4-8, and n is
The integer of 1-3.
4. a kind of preparation method of long-chain biological element marker according to claim 3 comprising:
(1) in pyridine solution, biotin and DMT-Cl is made to react to obtain intermediate product A；
(2) in anhydrous dimethyl formamide, intermediate product A is made to react to obtain intermediate product B with amino-hexanol；
(3) in anhydrous acetonitrile, intermediate product B is activated with chloro-carbonic acid p-nitrophenyl phenolic ester, is reacted later with amino-PEG3- alcohol
Intermediate product C1 is obtained, replaces intermediate product B to repeat the step again n-1 times with intermediate product C1, obtains intermediate product Cn,
Meaning wherein in the meaning with formula (I) of n is identical；
(4) in anhydrous acetonitrile, intermediate product Cn is made to react to obtain long-chain biological element label with phosphorus reagent under tetrazole effect
Object.
5. according to the method described in claim 4, wherein using two succinimidyl carbonates as catalysis in step (2)
Agent.
6. according to the method described in claim 4, wherein the phosphorus reagent in step (4) is 2- cyanoethyl-N, N, N ' the N isopropyl of '-four
Base phosphorous acid diamide.
7. long-chain biological element marker according to claim 1-3 is in preparation gene sequencing/detection probe
Purposes.
8. a kind of oligonucleotides comprising oligonucleotide sequence and long-chain biological according to claim 1-3 element
Marker, wherein the long-chain biological element marker is bound to the 5 ' terminal hydroxy group of oligonucleotide sequence.
9. a kind of preparation method of oligonucleotides comprising:
The step of (1 ') is by multiple nucleotide sequence synthetic oligonucleotides；With
(2 ') long-chain biological element marker combination step comprising use tetrazole admixture activation long-chain biological element marker, so
The oligonucleotide sequence 5 ' for obtaining it with step (1) holds the hydroxyl in the last one base to be reacted；Then oxygen is carried out
Change and DMT is gone to protect.
10. long-chain biological element marker according to claim 1-3 or few nucleosides according to claim 8
Purposes of the acid in two generations were sequenced.
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