USRE40557E1 - Methods for labeling nucleotides, labeled nucleotides and useful intermediates - Google Patents
Methods for labeling nucleotides, labeled nucleotides and useful intermediates Download PDFInfo
- Publication number
- USRE40557E1 USRE40557E1 US10/900,902 US90090299A USRE40557E US RE40557 E1 USRE40557 E1 US RE40557E1 US 90090299 A US90090299 A US 90090299A US RE40557 E USRE40557 E US RE40557E
- Authority
- US
- United States
- Prior art keywords
- spacer
- label
- nucleotide
- dna
- moiety
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims abstract description 88
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 55
- 239000002773 nucleotide Substances 0.000 title claims abstract description 49
- 238000002372 labelling Methods 0.000 title claims abstract description 47
- 239000000543 intermediate Substances 0.000 title description 6
- 125000006850 spacer group Chemical group 0.000 claims abstract description 46
- 125000005842 heteroatom Chemical group 0.000 claims abstract description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 36
- 229960002685 biotin Drugs 0.000 claims description 18
- 235000020958 biotin Nutrition 0.000 claims description 18
- 239000011616 biotin Substances 0.000 claims description 18
- 125000004432 carbon atom Chemical group C* 0.000 claims description 14
- -1 aliphatic diamine Chemical class 0.000 claims description 9
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- 238000005859 coupling reaction Methods 0.000 claims description 7
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 7
- 239000003446 ligand Substances 0.000 claims description 7
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical group CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 6
- 150000002367 halogens Chemical class 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- 229930024421 Adenine Natural products 0.000 claims description 4
- 108010090804 Streptavidin Proteins 0.000 claims description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 4
- 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 claims description 4
- 229960000643 adenine Drugs 0.000 claims description 4
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 4
- 229940104302 cytosine Drugs 0.000 claims description 4
- 108090001008 Avidin Proteins 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims 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 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 108010039918 Polylysine Proteins 0.000 claims description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 2
- 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 claims description 2
- 239000003638 chemical reducing agent Substances 0.000 claims description 2
- 125000003916 ethylene diamine group Chemical group 0.000 claims description 2
- IWBOPFCKHIJFMS-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl) ether Chemical compound NCCOCCOCCN IWBOPFCKHIJFMS-UHFFFAOYSA-N 0.000 claims description 2
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 2
- 229920000656 polylysine Polymers 0.000 claims description 2
- 230000002285 radioactive effect Effects 0.000 claims description 2
- 229940104230 thymidine Drugs 0.000 claims description 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 2
- 229940045145 uridine Drugs 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims 2
- 125000003118 aryl group Chemical group 0.000 claims 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims 1
- 239000000470 constituent Substances 0.000 claims 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 claims 1
- 229910052760 oxygen Inorganic materials 0.000 claims 1
- 239000001301 oxygen Substances 0.000 claims 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 claims 1
- 150000007944 thiolates Chemical group 0.000 claims 1
- 150000003058 platinum compounds Chemical group 0.000 abstract description 18
- 230000008901 benefit Effects 0.000 abstract description 11
- 230000000087 stabilizing effect Effects 0.000 abstract description 6
- 125000004429 atom Chemical group 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 64
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 63
- 239000000523 sample Substances 0.000 description 38
- 150000001875 compounds Chemical class 0.000 description 34
- 239000000243 solution Substances 0.000 description 28
- 239000003298 DNA probe Substances 0.000 description 19
- 239000012528 membrane Substances 0.000 description 18
- 125000005647 linker group Chemical group 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 239000013580 millipore water Substances 0.000 description 12
- 229910052697 platinum Inorganic materials 0.000 description 12
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 229940045985 antineoplastic platinum compound Drugs 0.000 description 10
- 238000009396 hybridization Methods 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 239000007858 starting material Substances 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 108020003215 DNA Probes Proteins 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- 229940012017 ethylenediamine Drugs 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 0 *[Pt]B Chemical compound *[Pt]B 0.000 description 7
- 238000004108 freeze drying Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 229910052709 silver Inorganic materials 0.000 description 6
- 239000004332 silver Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Substances [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000004677 Nylon Substances 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229920001778 nylon Polymers 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- QEIFSLUFHRCVQL-UHFFFAOYSA-N (5-bromo-4-chloro-1h-indol-3-yl) hydrogen phosphate;(4-methylphenyl)azanium Chemical compound CC1=CC=C(N)C=C1.C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QEIFSLUFHRCVQL-UHFFFAOYSA-N 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 230000004568 DNA-binding Effects 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000004816 latex Substances 0.000 description 3
- 229920000126 latex Polymers 0.000 description 3
- 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 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 125000004433 nitrogen atom Chemical group N* 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 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 3
- 239000000758 substrate Substances 0.000 description 3
- 241000972773 Aulopiformes Species 0.000 description 2
- LCXYLCUMZRNWDJ-UHFFFAOYSA-N C.C[Pt]NCN Chemical compound C.C[Pt]NCN LCXYLCUMZRNWDJ-UHFFFAOYSA-N 0.000 description 2
- RSJVGFDAEGHFNN-UHFFFAOYSA-N C[Pt](C)(C)NCN Chemical compound C[Pt](C)(C)NCN RSJVGFDAEGHFNN-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 241001428582 Human papillomavirus type 6 Species 0.000 description 2
- 241000343203 Lidia Species 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical group [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- FRBUGGTUSNODGZ-UHFFFAOYSA-N O=[N+]([O-])O[Pt]1(O[N+](=O)[O-])NCCN1 Chemical compound O=[N+]([O-])O[Pt]1(O[N+](=O)[O-])NCCN1 FRBUGGTUSNODGZ-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108020004518 RNA Probes Proteins 0.000 description 2
- 239000003391 RNA probe Substances 0.000 description 2
- 229910021612 Silver iodide Inorganic materials 0.000 description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- REGCCBIMYYRCSY-UHFFFAOYSA-N azanium;n,n-diethylethanamine;acetate Chemical compound [NH4+].CC([O-])=O.CCN(CC)CC REGCCBIMYYRCSY-UHFFFAOYSA-N 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 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 2
- 238000000151 deposition Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 150000004985 diamines Chemical class 0.000 description 2
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 2
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 150000003057 platinum Chemical class 0.000 description 2
- HRGDZIGMBDGFTC-UHFFFAOYSA-N platinum(2+) Chemical compound [Pt+2] HRGDZIGMBDGFTC-UHFFFAOYSA-N 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- OPCJOXGBLDJWRM-UHFFFAOYSA-N 1,2-diamino-2-methylpropane Chemical compound CC(C)(N)CN OPCJOXGBLDJWRM-UHFFFAOYSA-N 0.000 description 1
- JKFYKCYQEWQPTM-UHFFFAOYSA-N 2-azaniumyl-2-(4-fluorophenyl)acetate Chemical group OC(=O)C(N)C1=CC=C(F)C=C1 JKFYKCYQEWQPTM-UHFFFAOYSA-N 0.000 description 1
- NMUURUZMXPDLAB-UHFFFAOYSA-N 2-methylbutane-1,2-diamine Chemical compound CCC(C)(N)CN NMUURUZMXPDLAB-UHFFFAOYSA-N 0.000 description 1
- ACNUVXZPCIABEX-UHFFFAOYSA-N 3',6'-diaminospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(N)C=C1OC1=CC(N)=CC=C21 ACNUVXZPCIABEX-UHFFFAOYSA-N 0.000 description 1
- DEQPBRIACBATHE-FXQIFTODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-2-iminopentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCC(=N)C(=O)O)SC[C@@H]21 DEQPBRIACBATHE-FXQIFTODSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 206010059313 Anogenital warts Diseases 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- FBEIPJNQGITEBL-UHFFFAOYSA-J Cl[Pt](Cl)(Cl)Cl Chemical compound Cl[Pt](Cl)(Cl)Cl FBEIPJNQGITEBL-UHFFFAOYSA-J 0.000 description 1
- IROITDSZMKWTSF-UHFFFAOYSA-L Cl[Pt]1(Cl)NCCN1 Chemical compound Cl[Pt]1(Cl)NCCN1 IROITDSZMKWTSF-UHFFFAOYSA-L 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 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
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-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
- 238000004566 IR spectroscopy Methods 0.000 description 1
- RNJPWBVOCUGBGY-UHFFFAOYSA-J I[Pt](I)(I)I Chemical compound I[Pt](I)(I)I RNJPWBVOCUGBGY-UHFFFAOYSA-J 0.000 description 1
- ZHMALHXTAHEELB-UHFFFAOYSA-L I[Pt]1(I)NCCN1 Chemical compound I[Pt]1(I)NCCN1 ZHMALHXTAHEELB-UHFFFAOYSA-L 0.000 description 1
- 229910020427 K2PtCl4 Inorganic materials 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 206010027146 Melanoderma Diseases 0.000 description 1
- BAQMYDQNMFBZNA-UHFFFAOYSA-N N-biotinyl-L-lysine Natural products N1C(=O)NC2C(CCCCC(=O)NCCCCC(N)C(O)=O)SCC21 BAQMYDQNMFBZNA-UHFFFAOYSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- BAQMYDQNMFBZNA-MNXVOIDGSA-N biocytin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCC[C@H](N)C(O)=O)SC[C@@H]21 BAQMYDQNMFBZNA-MNXVOIDGSA-N 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000000484 continuous-wave nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- LMBWSYZSUOEYSN-UHFFFAOYSA-N diethyldithiocarbamic acid Chemical compound CCN(CC)C(S)=S LMBWSYZSUOEYSN-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229950004394 ditiocarb Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- WTOSNONTQZJEBC-UHFFFAOYSA-N erythrosin Chemical compound OC(=O)C1=CC=CC=C1C(C1C(C(=C(O)C(I)=C1)I)O1)=C2C1=C(I)C(=O)C(I)=C2 WTOSNONTQZJEBC-UHFFFAOYSA-N 0.000 description 1
- LMABILRJNNFCPG-UHFFFAOYSA-L ethane-1,2-diamine;platinum(2+);dichloride Chemical compound [Cl-].[Cl-].[Pt+2].NCCN LMABILRJNNFCPG-UHFFFAOYSA-L 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 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 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 239000008131 herbal destillate Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 231100001225 mammalian toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000000247 oncostatic effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 150000001282 organosilanes Chemical class 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 150000003141 primary amines Chemical group 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000000763 radioactive isotope labeling Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 229940045105 silver iodide Drugs 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/80—Fluorescent dyes, e.g. rhodamine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
Definitions
- the invention relates to methods for labeling nucleotides using linkers (linking moieties between labels and bio-organic molecules, which linkers are based on platinum compounds).
- Platinum (coordination) compounds have been considered interesting molecules for a very long time. For a review of these compounds and their uses we refer to Reedijk et al. (Structure and Bonding 67, p.53-89, 1987). Especially Cis-platinum has received a lot of attention as a possible anti-tumour drug.
- This anti-tumour reactivity of platinum compounds originates from their having at least two reactive groups (preferably cis-oriented towards each other), which make it possible to cross-link DNA molecules, thereby inhibiting the replication of these DNA molecules.
- the British patent application 2 148 891 discloses cis-platinum complexes, which are six-coordinated.
- the platinum is attached to two halogens or hydroxy groups, two additional halogens and to an ethylenediamine derived group, such as 1,2-diamino-2-methylpropane or 1,2-diamino-2-methylbutane.
- the complexes are said to have an excellent anti-tumor effect.
- the complexes comprise a diamine bidentate ligand and two 2-arylalkanoic acid or 3-aryl-2-oxoalkanoic acid ligands. These complexes are stated to have a strong growth inhibiting action on certain leukemia cells and are used for their oncostatic activity.
- U.S. Pat. No. 4,207,416 discloses ethylenediamine-platinum(II) 2,4-dioxopyrimidine complexes as having a high anti-tumor activity and low mammalian toxicity.
- a major drawback of these known methods is that they are not suitable for labeling all different nucleotides. For instance, Dale et al. reported that their covalent mercuration method leads to negative results for adenine, thymine and guanine bases. In some cases, for example when only a few residues of a certain nucleotide are present in a certain nucleic acid or when the terminating nucleotide residue of a nucleic acid has to be labeled, it is desired to have at one's disposal a method for labeling any nucleotide residue.
- the present invention provides such a method.
- the method for labeling nucleotides of the invention comprises the steps of:
- the linker may first be attached to the nucleotide and then to the spacer, or vice versa and the spacer may first be coupled to the label and then to the linker or vice versa.
- the reactive moiety of the spacer may be any reactive moiety that will enable the reaction between the spacer and the label in such a manner that a labeling moiety comprising a label and a spacer is formed, which labeling moiety is sufficiently stable.
- nucleotides can be incorporated in nucleic acid molecules.
- Modified nucleotides especially those wherein a (bulky) label is attached to the nucleotide, are often built-in into nucleic acids with a much lower efficiency.
- the methods according to the invention result in labeled nucleotides which are built-in into nucleic acids with a higher efficiency than the labeled nucleotides available to date. This is probably due to the selection of the spacers according to the invention in combination with the platinum-based linkers according to the invention.
- the label to be used according to the invention is not critical. In principle all labels which can be attached to a nucleotide and are employed to date can be used. These labels may be radioactive labels, enzymes (which need reaction with a substrate to be detected), specific binding pairs components such as avidin, streptavidin or biotin, biocytin, iminobiotin, colloidal dye substances, fluorochromes (rhodamin, etc.), reducing substances, (eosin, erythrosin, etc.), (coloured) latex sols, digoxigenin, metals (ruthenium), metal sols or other particulate sols (selenium, carbon and the like), dansyl lysin, Infra Red Dyes, coumarines (amino methyl coumarine), antibodies, protein A, protein G, etc.
- the invention has most benefits with bulkier labels such as biotin, avidin, streptavidin, digoxygenin or a functional equivalent thereof.
- the invention is not limited to nucleotides or nucleosides as such: derivatives and functional equivalents are also included.
- the usual nucleotides adenine, thymidine, cytosine, guanine and uridine are preferred.
- Especially the purines are preferred which have a very good incorporation rate.
- the electron donating moiety is an amine or a thiolate anion, which have both proven to be very successful. It was found that aromatic amines, such as imidazoles or purines, are capable of forming very strong bonds to platinum and thus are very suitable for use as the electron donating moiety.
- the spacer is an important aspect of the present invention; it provides the easiest coupling between label and linker.
- the nucleotide For avoiding steric hindrance in incorporation of the nucleotide into the nucleic acid it should at least be four atoms long, preferably it is at least four carbon atoms long and has at least one heteroatom in that carbon chain.
- a heteroatom confers a certain amount of rigidity on the spacer. This rigidity provides an additional assurance that steric factors will not obstruct a convenient linking of a nucleotide and a label. It is preferred that at least one heteroatom is an oxygen atom, which positively effects the hydrophilicity of the spacer.
- the spacer comprises no more than 20 carbon atoms in the chain, which is preferably an essentially non-branched chain, thus causing no steric hindrance. The reason for this will be clear.
- a highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, herein referred to as Dadoo.
- Dadoo is a very flexible compound with a distal primary amine group and a size that makes it very suitable for use as spacer according to the invention.
- Another highly preferred spacer of the invention is an oligolysine or a polylysine. Due to their structure and conformation, these molecules create the most convenient environment for an optimal interaction among the actual label, the nucleotide and the platinum.
- An additional advantage of the use of lysine chains as the spacer is, that by altering the number of lysine units in the chain, the optimal conditions for specific labels and nucleotides or nucleic acids can be attained. Given a certain application, the skilled person will easily determine how many lysine units are required for optimum results.
- An especially interesting labeling moiety comprising a label and a spacer, has the formula or the formula wherein X represents any stabilizing bridge, Z and Z′ represent a non-leaving ligand and n is an integer of from 2 to 10.
- linker-spacer-label-system, or labeling substance, with the labeling moiety of formula (II) or formula (III) has the formula or the formula wherein A, X, Z, Z′ and n have the above meanings.
- the non-leaving ligands Z and/or Z′ are preferably an NH 3 , NH 2 R, NHR 2 or NR 3 group, wherein R represents an alkyl group having from 1 to 6 carbon atoms, because these ligands have an even smaller leaving-group character than other non-leaving ligands.
- the linkers according to the invention preferably are platinum compounds wherein X represents an aliphatic diamine.
- X represents an aliphatic diamine.
- one or both of the nitrogen atoms of the aliphatic diamine are shielded.
- a suitable manner of shielding these nitrogen atoms consists of substitution with one or two alkyl groups of from 1 to 6 carbon atoms, preferably methyl groups. This is advantageous in that hydrogen bonding between the triphosphate group of a nucleotide and the stabilizing bridge is prevented.
- a diamine having 2-6 carbon atoms is used, most preferably an ethylene diamine group, which is well-known for its stabilizing effect on this class of platinum compounds.
- the linker has the formula wherein G represents hydrogen or an alkyl group of from 1 to 6 carbon atoms and A and B represent the same or different reactive moieties.
- the coupling or reactive moieties A and B are preferably the same and selected from the group consisting of NO 3 ⁇ , SO 3 ⁇ , Cl 31 , I ⁇ , or other halogens.
- the invention of course also encompasses a labeled nucleotide obtainable by a method as disclosed above.
- the invention encompasses a labeling substance for labeling nucleotides by a method as disclosed above.
- the labeling substance of the invention has the formula wherein X and A have the above meanings and the labeling moiety comprises a label and a spacer as described above.
- the labeling substances of the invention can also be used for labeling purposes other than labeling nucleotides. It was found that numerous (bio-) organic compounds, i.e. nearly every bio-organic molecule which contains an accessible sulphur or nitrogen atom, for example proteins, can be labeled with the platinum compounds of the invention.
- a great advantage of the invention arises from the use of the platinum compounds having formula (I) as linkers in the methods of preparing labeled nucleotides according to the invention. These linkers can be prepared by very convenient and reliable methods.
- the starting compounds disclosed for preparing labeled platinum compounds are platinum(II) (ethylenediamine)dichloride and platinum(II) (ethylenediamine)(Me 2 SO)Cl.
- the first one can be obtained commercially, the second one (the preferred one) must be synthesized.
- the Cl-ions are less readily substituted by a label or a nucleotide, respectively. In the latter case, the total nucleotide labeling time will be appreciably longer, up to several hours, instead of several minutes.
- the methods for preparing the linkers that are used in the method of labeling nucleotides according to the invention are based on the selection of suitable starting compounds of the formula PtE 4 wherein E is an electronegative group, preferably a halogen or NO 3 ⁇ or SO 3 ⁇ .
- E is an electronegative group, preferably a halogen or NO 3 ⁇ or SO 3 ⁇ .
- the reaction, which is described in the examples, of these starting compounds with e.g. ethylenediamine is very simple and efficient. Moreover, this reaction leads to very suitable symmetric intermediate compounds for producing labeled nucleotides.
- a major advantage of using these compounds is that when a stabilizing bridge for the resulting platinum compound has to be attached that no blocking reagents have to be employed. Another advantage is that the resulting intermediate compounds can again be labeled without the use of blocking agents.
- a very suitable intermediate compound according to the invention is platinum(II)(ethylenediamine)(NO 3 ) 2 .
- This substance can very easily be provided with a suitable spacer and a labeling group, resulting in labeling substances which can, through substitution of the remaining NO 3 -group be linked to a nucleotide quite easily.
- the methods for producing these compounds and the resulting compounds do not involve highly toxic substances such as DMSO.
- the intermediate compounds can be labeled with any suitable label (also known as marker) through a spacer as disclosed hereinabove.
- platinum compounds are of course also obtained with the present methods and compounds.
- Another advantage of the platinum compounds is that they can be detected more or less directly by using the platinum as a nucleus for depositing silver or other metal crystals.
- DNA or RNA molecules By binding the labeling substance to a nucleotide residue, DNA or RNA molecules, be it single stranded or otherwise, can be easily detected, but it also allows for the production of probes for hybridization techniques wherein unlabeled DNA/RNA molecules hybridize to the labeled probe.
- the platinum linker labeled nucleotides hardly interfere with the hybridization, if at all. Also, this technique obviates the use of modified nucleotides in preparing probes.
- Nucleotides modified in accordance with the practices of this invention and oligo- and polynucleotides into which the modified nucleotides have been incorporated or oligo- and polynucleotides that have been directly modified using these novel platinum compounds may be used as probes in biomedical research, clinical diagnostics and recombinant DNA technology.
- the filtrate should be clear.
- Example 1A Repeat the entire procedure of Example 1A, but use N,N,N′, N′-tetramethylethylenediamine instead of ethylenediamine.
- Dissolve Pt(en)(NO 3 ) 2 (18.2 mg, 0.048 mmol) in 10 ml of Millipore water and heat until dissolving.
- Dissolve BioDadoo (20 mg, 0.053 mmol, purchased from Boehringer Mannheim) in 5 ml of Millipore water. Add the two solutions together and adjust the pH to 8 by 0.1 N NaOH, react for at least 3 hours at 50° C. Isolate the end product by freeze drying.
- Dissolve Pt(tmen)(NO 3 ) 2 35 mg, 0.08 mmol
- Dissolve BioDadoo 32 mg, 0.085 mmol
- Dissolve Pt(en)(NO 3 ) 2 (5 mg, 0.013 mmol) in 5 ml of Millipore water and heat until dissolving.
- Dissolve DigDadoo (9 mg, 0.016 mmol, purchased from Boehringer Mannheim) in 5 ml of Millipore water. Add the two solutions together and react for at least 4 hours at 50° C. Isolate the end product by freeze drying.
- the labeling method has been used to label DNA probes with Biotin for In Situ Hybrization (ISH).
- ISH Biotin for In Situ Hybrization
- labeling procedures including the protocols and data for quality control procedures are presented.
- Biotin labeling a plasmid cloned total DNA of Human Papilloma Virus type 6 (HPV-6, 40% GC basepairs) was used.
- Plasmid DNA was transferred into E. coli (C-600) and plated onto ampicillin containing LB plates Single colonies were grown overnight in large culture. Plasmid DNA was isolated according to the method of Birnboim and Doly 1 , purified by Sepharose C1-2B columnchromatography (Pharmacia) and checked for inserts by restriction-enzyme-analyses. Plasmid DNA concentration was determined by A260/280 absorbtion. After ethanol precipitation the DNA was reconstituted in 10 mM TRIS/HCl pH 7.2, 0.3 mM EDTA to a final concentration of 1 ⁇ g/ ⁇ l (batch# 150894). Subsequently this DNA was sonicated (Soniprep 150., MSE) for 3 times 10 minutes (amplitude 5 microns) on ice.
- the size of the resulting DNA fragments was determined by 2% agarose gel electrophoresis and found to be in between 200-400 basepairs (batch# 051094).
- Plasmid HPV-6 DNA was labeled with BioDadoo-ULS by mixing the following reagents:
- the detection limit of the biotin probe of the invention was determined by direct spot blot and reversed filterhybridization according to the following protocols:
- HPV-6 probe (batch# 061094) labeled with biotin according to the invention was 10-fold serially diluted into spot buffer comprising 900 mM sodium chloride, 90 mM sodium citrate and 200 ⁇ g/ml single stranded salmon sperm DNA giving a dilution series varying from 1000-0.1 pg biotin probe per ⁇ l. 1 ⁇ l spots were applied onto nitrocellulose membrane and incubated for 2 hours at 80° C. to blind the DNA. The biotin probe was visualized using a streptavidin-alkaline phosphatase conjugate (Sigma) combined with a NBT/BCIP precipitating substrate solution (Sigma) according to the following protocol:
- the detection limit of the biotin DNA probe according to the invention was found to be less than 1 pg.
- HPV-6 DNA (batch# 051094) was1 in 10 diluted in 0.1N NaOH, incubated at 100° C. for 5 minutes and directly placed on ice for 5 minutes to make DNA single stranded.
- HPV-6 DNA probe that was labeled with biotin according to the invention was diluted in 5 ⁇ SSPE 0.5% SDS solution to yield a concentration of 200 ng/ml.
- This solution was incubated for 5 minutes at 100° C. and placed directly on ice for 5 minutes.
- Nylon membranes containing target DNA were soaked in 2 ⁇ SSC for 5 minutes and subsequently incubated with the single stranded probe solution for 2 hours at 37° C.
- biotin probe of the invention was visualized by performing the same protocol as described in the direct spot blot method.
- the detection limit of the biotin probe according to the invention was found to be less than 10 pg.
- test material consisted of 6 ⁇ m paraffin sections of a HPV-6 posotive cervical condyloma mounted on organosilane coated glass slides.
- Probe solution consisted of biotin HPV-6 probe DNA labeled according to the invention (batch# 061094) in a concentration of 2 ng/ ⁇ l dissolved in hybridization mixture comprising 0.6M NaCl, 0.06M sodium citrate, 35% formamid, 10% dextransulphate, 2,5 ⁇ Denhardts and 10 ⁇ g/ml single stranded salmon sperm DNA.
- the present method is very well suited for research, routine and for industrial production of labeled nucleic acids, as the method is fast and easy to perform, very sensitive, and does not include any enzymatic step, which makes it highly reproducible and fitted for an overall low cost production.
- the method of the invention offers a useful alternative equaling conventional non-isotopic labeling methods.
- the LIDIA technique enables the quantitative analysis of small amounts of DNA (or RNA) e.g. after a PCR amplification of the starting material.
- the technique is sensitive and specific, due to the use of specific DNA(RNA) probes in accordance with the invention and easy to perform, because of the quick DNA(RNA) Pt-labeling steps of the invention.
- the technique uses fast Pt labeling compounds of the invention to label DNA(RNA) probes
- An immobilized DNA probe can be used to catch specific target molecules in a sample by using a hybridization technique. Detection of formed hybrids can be done by using different techniques, e.g. a second labeled DNA probe can be used to hybridize with a different site on the target DNA molecule to form a sandwich hybrid. The label can then be detected by using state of the art immunological detection and colouring techniques.
- a volume containing (amplified) detectable DNA(RNA) is directly labeled according to the protocol in accordance with the invention.
- a second step is performed in a microtiter plate precoated with a target specific probe. Incubation is allowed to the formation of stable “Labelled target” and probe hybrids.
- the direct labeling of target molecules enables the omission of laborious double hybridization techniques where one probe is used to catch the target and another labeled probe is used to detect the immobilized target.
- the probes are covalently linked to the microtiter plate to the surface of the wells.
- the second incubation step has the character of a liquid hybridisation and therefore can be performed very rapidly. This is one of the main innovative features of this approach to quantitive DNA hybridisation techniques.
- Both for the immobilization of DNA probes or DNA targets and for the labeling of DNA probes and targets the newly developed Pt system can be used.
- These two DNA linking techniques can be combined into one assay where both the “catcher” and the “detector” are linked to a second substance (either a detectable group like biotin, digoxigenin or a carrier surface like a plastic stick, microtiter plate or a membrane).
- the DNA dipstick technique enables the qualitative and semi-quantitative analysis of small amounts of DNA(or RNA) e.g. after a PCR amplification or freely present in samples of body fluids (blood, urine, sweat etc.)
- the technique is sensitive and specific, due to the use of specific DNA(RNA) probes and easy to perform because of the quick DNA(RNA) Pt-labeling steps according to the invention.
- the universal labeling characteristics of the newly developed Pt label can be used in 3 ways to achieve a bound DNA(RNA) molecule.
- a DNA probe can be labeled with the newly developed Pt labeling compound.
- This labeled probe can then be used to detect preformed hybrids on a membrane formed between the target DNA sequence and a primary probe. It is essential in this method that the primary probe recognizes a different sequence on the target than the secondary Pt labeled probe. In practice, this can be achieved for instance with RNA hybridization where a POLY T probe is used as a primary probe to immobilize all RNA (recognizable by its POLY A tails) to a membrane.
- the second approach differs slightly in that in this case the target can be labeled in the test sample fluid, because of the fast and very specific Pt labeling characteristics.
- a procedure like this would comprise a catch of the labeled target with an immobilized specific unlabeled DNA probe on a suitable membrane. Hence a dipstick version for DNA/RNA applications.
- a non-labeled Pt compound that is a Pt compound with 2 free binding sites
- carriers plastic, membrane, microtiter plates etc.
- the use of the Pt compound of the invention in latex or hydrosol assays is particularly interesting.
- the compound enables the coupling of DNA molecules to small particles.
- the DNA molecules can be hybridized to target material.
- a positive reaction is visualized by an agglutination of the particles, due to crosslinking of the DNA hybrid particle compounds.
- a test like this can be made quantative, the rate of agglutination can be tuned and measured at a specific wavelength.
- gold particles have the intrinsic characteristic that a shift in optimal wavelength occurs after agglutination.
- Platinated DNA/RNA probe can be employed in hydridisation methods to detect DNA/RNA sequences in sample material.
- the introduction of a platinum compound at the site of the target enables the deposition of Ag molecules in a chemical reaction especially designed to reduce ionic silver to metallic silver.
- a decomposition of metallic silver(black) occurs due to the catalytic effect of the Pt nucleus.
- Ionic silver is reduced by a reducing agent (e.g. Na-borohydrid, Hydrochinon) in solution.
- a reducing agent e.g. Na-borohydrid, Hydrochinon
- the amount of silver deposited on the Pt is proportional to the length of the enhancement incubation.
- Visualisation of a non-visible Pt nucleus can be accomplished by the empirical observation of the appearace of a black spot in the test sample.
- the black spots indicate the site of specific probes binding and thus the site of specific target location.
- the technique enables a quick and easy diagnostic procedure for the detection of various microorganisms and gene translocations/abnormalities.
Abstract
Description
- reacting a reactive moiety of a linker of the formula
- wherein X represents any stabilizing bridge and wherein A and B represent the same or different reactive moieties, with an electron donating moiety of a spacer, which spacer comprises a chain having at least four atoms and at least one heteroatom in the chain, which spacer further comprises said electron donating moiety at one end of the chain and a reactive moiety at the other end of the chain;
- reacting the reactive moiety of said spacer with a label;
- reacting the other reactive moiety of said linker with a nucleotide.
or the formula
wherein X represents any stabilizing bridge, Z and Z′ represent a non-leaving ligand and n is an integer of from 2 to 10.
or the formula
wherein A, X, Z, Z′ and n have the above meanings.
wherein G represents hydrogen or an alkyl group of from 1 to 6 carbon atoms and A and B represent the same or different reactive moieties.
wherein X and A have the above meanings and the labeling moiety comprises a label and a spacer as described above. Of course the labeling substances of the invention can also be used for labeling purposes other than labeling nucleotides. It was found that numerous (bio-) organic compounds, i.e. nearly every bio-organic molecule which contains an accessible sulphur or nitrogen atom, for example proteins, can be labeled with the platinum compounds of the invention.
- (a) reacting a compound having the structure:
- with KI in a suitable solvent under suitable conditions so as to form a iodated platinum compound having the structure:
- (b) reacting said iodated platinum compound with ethylene-diamine in a suitable solvent so as to form a diethylene-amine iodated platinum compound and represented by the formula Pt(en)I2 and having the structure:
- (c) reacting said compound with AgNO3, the reaction being carried out in a suitable solvent, under suitable conditions so as to form a compound having the structure:
- (d) reacting said compound with KCl in a suitable solvent under suitable conditions so as to form a compound having the structure:
- (e) reacting said compound with AgNO3 in a suitable solvent, under suitable conditions so as to form a compound having the structure:
- (f) recovering said compound as modified platinum starting compound for the synthesis of hapten-bound Pt(en) compounds for use as DNA and/or RNA label.
plasmid HPV-6 DNA (batch #051094) | 5 | μl (1 μg/μl) | ||
BioDadoo-ULS labeling reagent | 8 | μl (1 μg/μl) | ||
(batch #BX940830) | ||||
Demineralised water (<0.2/μS/cm) | 37 | μl | ||
The 50 μl reaction mixture was incubated for 15 minutes at 85° C.
-
- Membranes were soaked in TBS solution containing 0.5% TWEEN20 (TBST) for 5 minutes
- Membranes were incubated with Strep-AP (3 DEA U/ml) in TBST for 15 minutes at 37° C.
- NC-membranes were washed 3 times 5 minutes in TBS solution followed by a 5 minute wash step in demineralised water.
- Membranes were incubated with NBT/BCIP substrate solution for 15 minutes at 37° C., subsequently washed in demineralised water and air dried.
Results
- 1 Paraffin sections were dewaxed in 3 changes of xylene and hydrated in graded ethanol.
- 2 Sections were rinsed in TBST for 5 minutes.
- 3 Sections were digested in 0.25% pepsin in 0.1N HCl for 30 minutes at 37° C., dehydrated in graded ethanol and air dried.
- 4 μl of probe solution was applied to a section and covered with a coverslip.
- 5 Slides were placed on a hot plate set at 95° C. for 5 minutes to denature probe and target DNA simultaneously.
- 6 Hybridization was performed by placing the slides in a humidified chamber at 37° C. for 2 hours.
- 7 Coverslips were removed and slides were washed in 3 changes of 15 mM NaCl, 1.5 mM sodium citrate and 5% formamid for 10 minutes at 37° C.
- 8 Slides were rinsed in TBST.
- 9 Sections were incubated with Streptavidin AP conjugate (3DEA U/ml in TBST) for 15 minutes at 37° C.
- 10 Slides were washed in TBST (3×) and demineralised water (1×) for 5 minutes.
- 11 Sections were incubated with NBT/BCIP substrated solution for 15 minutes at 37° C.
- 12 Slides were washed in demineralised water (3×) for 1 minute and sections were mounted in glycerol/gelatin.
Results
- 1. Maniatis T., Sambrook J., Fritsch E. F., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press, ISBN 0-8769-309-6.
- 2. Keller G. H., Manak M. M., DNA probes, Stockton Press, ISBN 0-333-47659-X.
Applications
1. The Use of Pt-DNA Linkers of the Invention in the So Called LIDIA Technique: Linked DNA Immuno Assay
- 1. Linking DNA probes molecules to a surface by using a Pt compound in accordance with the invention which crosslink DNA molecules irreversibly to plastic, nylon or nitrocellulose. Detection of DNA targets can then be accomplished by using classically labeled DNA/RNA probes.(nick translation or chemical modification, random priming)
- 2. Linking a detectable group to the DNA, to render a DNA molecule into a so-called DNA probe. Binding of DNA compounds to a surface can then be accomplished by using classic techniques known to science (covalent linking to specially treated microtiter plates, baking of DNA molecules onto nitrocellulose or binding of DNA molecules to nylon membranes.
- 3. A combination of techniques 1 and 2
Approach 1
- 1. It can be used to attach a detectable marker group to a polynucleotide sequence.
- 2. It can be used to attach polynucleotide sequences irreversibly to a solid phase (plastic, membranes, latex beads, hydrosols or microtiter plate wells).
- 3. A combination of 1 and 2
ad 1
Claims (28)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96202792 | 1996-10-08 | ||
PCT/NL1997/000559 WO1998015564A1 (en) | 1996-10-08 | 1997-10-08 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
Publications (1)
Publication Number | Publication Date |
---|---|
USRE40557E1 true USRE40557E1 (en) | 2008-10-28 |
Family
ID=8224469
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/402,707 Ceased US6338943B1 (en) | 1996-10-08 | 1997-10-08 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
US10/900,902 Expired - Lifetime USRE40557E1 (en) | 1996-10-08 | 1997-10-08 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
US10/047,874 Expired - Lifetime US6797818B2 (en) | 1996-10-08 | 2002-01-14 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
US10/950,985 Expired - Fee Related US7217813B2 (en) | 1996-10-08 | 2004-09-27 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/402,707 Ceased US6338943B1 (en) | 1996-10-08 | 1997-10-08 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/047,874 Expired - Lifetime US6797818B2 (en) | 1996-10-08 | 2002-01-14 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
US10/950,985 Expired - Fee Related US7217813B2 (en) | 1996-10-08 | 2004-09-27 | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
Country Status (12)
Country | Link |
---|---|
US (4) | US6338943B1 (en) |
EP (1) | EP0937090B1 (en) |
JP (2) | JP4981197B2 (en) |
AT (1) | ATE221894T1 (en) |
AU (1) | AU726692B2 (en) |
CA (1) | CA2267133C (en) |
DE (1) | DE69714600T2 (en) |
DK (1) | DK0937090T3 (en) |
ES (1) | ES2182121T3 (en) |
NZ (1) | NZ334803A (en) |
PT (1) | PT937090E (en) |
WO (1) | WO1998015564A1 (en) |
Families Citing this family (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0937090B1 (en) * | 1996-10-08 | 2002-08-07 | Kreatech Biotechnology B.V. | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
US6458373B1 (en) | 1997-01-07 | 2002-10-01 | Sonus Pharmaceuticals, Inc. | Emulsion vehicle for poorly soluble drugs |
EP1006199A1 (en) | 1998-12-03 | 2000-06-07 | Kreatech Biotechnology B.V. | Applications with and methods for producing selected interstrand crosslinks in nucleic acid |
WO2001029265A1 (en) * | 1999-10-15 | 2001-04-26 | Ventana Medical Systems, Inc. | Method of detecting single gene copies in-situ |
US6376669B1 (en) * | 1999-11-05 | 2002-04-23 | 3M Innovative Properties Company | Dye labeled imidazoquinoline compounds |
GB0016836D0 (en) | 2000-07-07 | 2000-08-30 | Lee Helen | Improved dipstick assays (1) |
DE60213387D1 (en) | 2001-03-02 | 2006-09-07 | Stratagene California | COMPOSITIONS AND METHODS OF PLATINUM COMPOUNDS FOR NUCLEIC ACID MARKING |
ATE376675T1 (en) * | 2001-05-28 | 2007-11-15 | Kreatech Biotech Bv | A METHOD FOR DIFFERENTIAL MARKING USING PLATINUM COMPLEXES |
US7166478B2 (en) * | 2002-03-12 | 2007-01-23 | Enzo Life Sciences, Inc., C/O Enzo Biochem, Inc. | Labeling reagents and labeled targets, target labeling processes and other processes for using same in nucleic acid determinations and analyses |
JP2005538735A (en) * | 2002-09-17 | 2005-12-22 | パーキネルマー ラス インコーポレイテッド | Real-time detection method of nucleic acid reaction |
US20050028450A1 (en) * | 2003-08-07 | 2005-02-10 | Wen-Qing Xu | CMP slurry |
US20060281100A1 (en) * | 2005-06-14 | 2006-12-14 | Shen Gene G | Thiotriphosphate nucleotide dye terminators |
EP1745802A1 (en) | 2005-07-20 | 2007-01-24 | Kreatech Biotechnology B.V. | Method of conjugating therapeutic compounds to cell targeting moieties via metal complexes |
JP5256026B2 (en) * | 2006-04-27 | 2013-08-07 | 株式会社ワンセル | Nucleotide-transition metal complex catalyst |
US20090215643A1 (en) | 2007-07-26 | 2009-08-27 | Cellay, Llc | Highly Visible Chromosome-Specific Probes and Related Methods |
KR101072900B1 (en) * | 2007-12-04 | 2011-10-17 | 주식회사 파나진 | Method for selective labeling and detection of target nucleic acids using immobilized peptide nucleic acid probes |
US10668162B2 (en) | 2012-01-06 | 2020-06-02 | Linxis B.V. | Method for preparing cell targeting conjugates, and the complexes obtained |
CN109030698B (en) * | 2018-08-06 | 2020-09-04 | 泰山医学院 | Method for detecting purity of solid sodium ferbamate by liquid chromatography |
CN108760961B (en) * | 2018-08-06 | 2020-09-22 | 青岛中科荣达新材料有限公司 | Method for detecting purity of solid potassium fomesate by liquid chromatography |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2148891A (en) * | 1983-10-19 | 1985-06-05 | Nippon Kayaku Kk | Platinum-diamine complexes |
EP0282672A1 (en) * | 1987-02-19 | 1988-09-21 | Nippon Kayaku Kabushiki Kaisha | Novel platinum complexes |
WO1992001699A1 (en) * | 1990-07-19 | 1992-02-06 | Stichting Klinische Research Academisch | Pt-CONTAINING COMPOUND, PROCESS FOR ITS PREPARATION, AND APPLICATION OF SUCH COMPOUNDS |
WO1996035696A1 (en) * | 1995-05-09 | 1996-11-14 | Kreatech Biotechnology B.V. | Methods for the production of platinum-based linkers between labels and bio-organic molecules, for labelling bio-organic molecules, for detecting biological substances of interest and diagnostic test kits |
US5985566A (en) * | 1990-07-19 | 1999-11-16 | Kreatech Diagnostics | Platinum-containing compounds, methods for their preparation and applications thereof |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4207416A (en) * | 1974-09-05 | 1980-06-10 | Engelhard Minerals & Chemicals Corporation | Ethylenediamineplatinum(II) 2,4-dioxopyrimidine complexes |
ATE40572T1 (en) * | 1985-01-10 | 1989-02-15 | Molecular Diagnostics Inc | PHOTOCHEMICAL METHOD FOR LABELING NUCLEIC ACIDS FOR DETECTION IN HYBRIDISATION SAMPLES. |
WO1989004317A1 (en) * | 1987-11-06 | 1989-05-18 | Sagami Chemical Research Center | Platinum (ii) complexes |
US5552541A (en) * | 1990-06-20 | 1996-09-03 | Beckman Instruments, Inc. | Haptenic probes for detecting capture polynucleotides |
CA2134239C (en) * | 1992-06-09 | 2004-11-23 | Donald B. Axworthy | Pretargeting methods and compounds |
JPH0698797A (en) * | 1992-09-22 | 1994-04-12 | Toyobo Co Ltd | Method for detecting nucleic acid |
DE4344742A1 (en) * | 1993-06-09 | 1994-12-15 | Boehringer Mannheim Gmbh | Method for the immobilization of nucleic acids |
DE4326466A1 (en) * | 1993-08-06 | 1995-02-09 | Boehringer Mannheim Gmbh | Infrared dye-labeled nucleotides and their use in nucleic acid detection |
EP0937090B1 (en) * | 1996-10-08 | 2002-08-07 | Kreatech Biotechnology B.V. | Methods for labeling nucleotides, labeled nucleotides and useful intermediates |
-
1997
- 1997-10-08 EP EP97943223A patent/EP0937090B1/en not_active Expired - Lifetime
- 1997-10-08 US US09/402,707 patent/US6338943B1/en not_active Ceased
- 1997-10-08 DK DK97943223T patent/DK0937090T3/en active
- 1997-10-08 ES ES97943223T patent/ES2182121T3/en not_active Expired - Lifetime
- 1997-10-08 US US10/900,902 patent/USRE40557E1/en not_active Expired - Lifetime
- 1997-10-08 CA CA002267133A patent/CA2267133C/en not_active Expired - Lifetime
- 1997-10-08 PT PT97943223T patent/PT937090E/en unknown
- 1997-10-08 AT AT97943223T patent/ATE221894T1/en active
- 1997-10-08 AU AU44746/97A patent/AU726692B2/en not_active Expired
- 1997-10-08 JP JP51741498A patent/JP4981197B2/en not_active Expired - Lifetime
- 1997-10-08 DE DE69714600T patent/DE69714600T2/en not_active Expired - Lifetime
- 1997-10-08 WO PCT/NL1997/000559 patent/WO1998015564A1/en active IP Right Grant
- 1997-10-08 NZ NZ334803A patent/NZ334803A/en not_active IP Right Cessation
-
2002
- 2002-01-14 US US10/047,874 patent/US6797818B2/en not_active Expired - Lifetime
-
2004
- 2004-09-27 US US10/950,985 patent/US7217813B2/en not_active Expired - Fee Related
-
2010
- 2010-01-29 JP JP2010019643A patent/JP2010166914A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2148891A (en) * | 1983-10-19 | 1985-06-05 | Nippon Kayaku Kk | Platinum-diamine complexes |
EP0282672A1 (en) * | 1987-02-19 | 1988-09-21 | Nippon Kayaku Kabushiki Kaisha | Novel platinum complexes |
WO1992001699A1 (en) * | 1990-07-19 | 1992-02-06 | Stichting Klinische Research Academisch | Pt-CONTAINING COMPOUND, PROCESS FOR ITS PREPARATION, AND APPLICATION OF SUCH COMPOUNDS |
US5985566A (en) * | 1990-07-19 | 1999-11-16 | Kreatech Diagnostics | Platinum-containing compounds, methods for their preparation and applications thereof |
WO1996035696A1 (en) * | 1995-05-09 | 1996-11-14 | Kreatech Biotechnology B.V. | Methods for the production of platinum-based linkers between labels and bio-organic molecules, for labelling bio-organic molecules, for detecting biological substances of interest and diagnostic test kits |
Also Published As
Publication number | Publication date |
---|---|
US6797818B2 (en) | 2004-09-28 |
DE69714600T2 (en) | 2003-03-27 |
EP0937090A1 (en) | 1999-08-25 |
AU726692B2 (en) | 2000-11-16 |
DE69714600D1 (en) | 2002-09-12 |
NZ334803A (en) | 2000-05-26 |
US6338943B1 (en) | 2002-01-15 |
DK0937090T3 (en) | 2002-12-02 |
JP2010166914A (en) | 2010-08-05 |
PT937090E (en) | 2002-12-31 |
CA2267133A1 (en) | 1998-04-16 |
ES2182121T3 (en) | 2003-03-01 |
US20020160396A1 (en) | 2002-10-31 |
EP0937090B1 (en) | 2002-08-07 |
US7217813B2 (en) | 2007-05-15 |
CA2267133C (en) | 2006-07-18 |
ATE221894T1 (en) | 2002-08-15 |
AU4474697A (en) | 1998-05-05 |
WO1998015564A1 (en) | 1998-04-16 |
JP2001503742A (en) | 2001-03-21 |
US20050118621A1 (en) | 2005-06-02 |
JP4981197B2 (en) | 2012-07-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
USRE40557E1 (en) | Methods for labeling nucleotides, labeled nucleotides and useful intermediates | |
US5985566A (en) | Platinum-containing compounds, methods for their preparation and applications thereof | |
US5449767A (en) | Modified polynucleotides and methods of preparing same | |
EP0063879B1 (en) | Modified nucleotides and methods of preparing and using same | |
EP1019420B1 (en) | Methods for the production of platinum-based linkers between labels and bio-organic molecules, for labelling bio-organic molecules, for detecting biological substances of interest | |
US4822731A (en) | Process for labeling single-stranded nucleic acids and hybridizaiton probes | |
EP0097373B1 (en) | Modified labeled nucleotides and polynucleotides and methods of preparing, utilizing and detecting same | |
US4617261A (en) | Process for labeling nucleic acids and hybridization probes | |
EP0329198B1 (en) | Base modified oligo- and polynucleotides and methods of preparing and using same | |
CA2286668C (en) | Trans-platinum compound, and diagnostic kit | |
EP0973785B1 (en) | Trans-platinum compound, and diagnostic kit | |
JPS63130599A (en) | Modified nucleotide | |
MXPA99003239A (en) | Methods for labeling nucleotides, labeled nucleotides and useful intermediates | |
US4983727A (en) | Palladium mediated reaction of organic disulfides with mercurated nucleic acid components | |
MXPA99009189A (en) | Trans-platinum compound, and diagnostic kit |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: KREATECH BIOTECHNOLOGY B.V., NETHERLANDS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HOUTHOFF, HENDRIK JAN;REEDIJK, JAN;JELSMA, TINKA;AND OTHERS;REEL/FRAME:010692/0156 Effective date: 19990921 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
RF | Reissue application filed |
Effective date: 20040728 |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
CC | Certificate of correction | ||
FPAY | Fee payment |
Year of fee payment: 8 |
|
FPAY | Fee payment |
Year of fee payment: 12 |
|
FEPP | Fee payment procedure |
Free format text: PAT HOLDER NO LONGER CLAIMS SMALL ENTITY STATUS, ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: STOL); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
AS | Assignment |
Owner name: PRIMOSA INTERNATIONAL N.V., NETHERLANDS ANTILLES Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KREATECH BIOTECHNOLOGY B.V.;REEL/FRAME:036383/0293 Effective date: 20150326 Owner name: LEICA BIOSYSTEMS NEWCASTLE LTD., UNITED KINGDOM Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PRIMOSA INTERNATIONAL N.V.;REEL/FRAME:036383/0358 Effective date: 20150326 |