GB2169605A - DNA synthesis - Google Patents
DNA synthesis Download PDFInfo
- Publication number
- GB2169605A GB2169605A GB08530915A GB8530915A GB2169605A GB 2169605 A GB2169605 A GB 2169605A GB 08530915 A GB08530915 A GB 08530915A GB 8530915 A GB8530915 A GB 8530915A GB 2169605 A GB2169605 A GB 2169605A
- Authority
- GB
- United Kingdom
- Prior art keywords
- dna
- endorphin
- gene
- long chain
- synthesis
- 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.)
- Granted
Links
- 230000006820 DNA synthesis Effects 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 31
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 13
- 238000010532 solid phase synthesis reaction Methods 0.000 claims abstract description 5
- 150000005691 triesters Chemical class 0.000 claims abstract description 4
- 239000005289 controlled pore glass Substances 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 44
- 108090000623 proteins and genes Proteins 0.000 description 21
- 108010049140 Endorphins Proteins 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 13
- 238000003786 synthesis reaction Methods 0.000 description 13
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 102000009025 Endorphins Human genes 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 102000053602 DNA Human genes 0.000 description 8
- 108020003215 DNA Probes Proteins 0.000 description 7
- 239000003298 DNA probe Substances 0.000 description 7
- 102100038796 E3 ubiquitin-protein ligase TRIM13 Human genes 0.000 description 6
- 101000664589 Homo sapiens E3 ubiquitin-protein ligase TRIM13 Proteins 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 108010041395 alpha-Endorphin Proteins 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 5
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- DNWBUCHHMRQWCZ-GUBZILKMSA-N Lys-Ser-Gln Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DNWBUCHHMRQWCZ-GUBZILKMSA-N 0.000 description 4
- DEGCBBCMYWNJNA-RHYQMDGZSA-N Thr-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O DEGCBBCMYWNJNA-RHYQMDGZSA-N 0.000 description 4
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 4
- 238000009833 condensation Methods 0.000 description 4
- 230000005494 condensation Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- PVNNAAWVJPIJHS-CQJMVLFOSA-N (2s)-2-[[(2s)-2-[[2-[[2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-4-methylsulfanylbutanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 PVNNAAWVJPIJHS-CQJMVLFOSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010022337 Leucine Enkephalin Proteins 0.000 description 2
- CULGJGUDIJATIP-STQMWFEESA-N Met-Tyr-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 CULGJGUDIJATIP-STQMWFEESA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 102100029563 Somatostatin Human genes 0.000 description 2
- BQBCIBCLXBKYHW-CSMHCCOUSA-N Thr-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@@H]([NH3+])[C@@H](C)O BQBCIBCLXBKYHW-CSMHCCOUSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 238000000211 autoradiogram Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229940039227 diagnostic agent Drugs 0.000 description 2
- 239000000032 diagnostic agent Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 108010047064 gamma-Endorphin Proteins 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 229960000553 somatostatin Drugs 0.000 description 2
- YCICLRBTJMLLGG-UHFFFAOYSA-N (2-chlorophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=CC=C1Cl YCICLRBTJMLLGG-UHFFFAOYSA-N 0.000 description 1
- ZDSRFXVZVHSYMA-CMOCDZPBSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-carboxybutanoyl]amino]pentanedioic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 ZDSRFXVZVHSYMA-CMOCDZPBSA-N 0.000 description 1
- RNHDAKUGFHSZEV-UHFFFAOYSA-N 1,4-dioxane;hydrate Chemical compound O.C1COCCO1 RNHDAKUGFHSZEV-UHFFFAOYSA-N 0.000 description 1
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101800005049 Beta-endorphin Proteins 0.000 description 1
- 102400000748 Beta-endorphin Human genes 0.000 description 1
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 102400000113 Calcitonin Human genes 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 201000008892 GM1 Gangliosidosis Diseases 0.000 description 1
- 102100040004 Gamma-glutamylcyclotransferase Human genes 0.000 description 1
- 102400000921 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 101000886680 Homo sapiens Gamma-glutamylcyclotransferase Proteins 0.000 description 1
- 101000632994 Homo sapiens Somatostatin Proteins 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- -1 N-Benzoyladenosyl Chemical group 0.000 description 1
- CFFXTXXQXWZQNT-UHFFFAOYSA-N N1=CC=CC=C1.CN(C(N(C)C)=N)C Chemical compound N1=CC=CC=C1.CN(C(N(C)C)=N)C CFFXTXXQXWZQNT-UHFFFAOYSA-N 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ZYPUVVZACMVNBV-UHFFFAOYSA-N S(=O)(=O)=C1[CH-]N(N=N1)[N+](=O)[O-].C1(=CC(=CC(=C1)C)C)C Chemical compound S(=O)(=O)=C1[CH-]N(N=N1)[N+](=O)[O-].C1(=CC(=CC(=C1)C)C)C ZYPUVVZACMVNBV-UHFFFAOYSA-N 0.000 description 1
- 108010086019 Secretin Proteins 0.000 description 1
- 102100037505 Secretin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- WOPZMFQRCBYPJU-NTXHZHDSSA-N beta-endorphin Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)C1=CC=CC=C1 WOPZMFQRCBYPJU-NTXHZHDSSA-N 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 238000006642 detritylation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- OENLEHTYJXMVBG-UHFFFAOYSA-N pyridine;hydrate Chemical compound [OH-].C1=CC=[NH+]C=C1 OENLEHTYJXMVBG-UHFFFAOYSA-N 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229960002101 secretin Drugs 0.000 description 1
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 108010068794 tyrosyl-tyrosyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- NXSIJWJXMWBCBX-NWKQFZAZSA-N α-endorphin Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 NXSIJWJXMWBCBX-NWKQFZAZSA-N 0.000 description 1
- GASYAMBJHBRTOE-WHDBNHDESA-N γ-endorphin Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 GASYAMBJHBRTOE-WHDBNHDESA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method of synthesizing long chain DNA, in which blocks having 4 to 8 base sequences are purely chemically ligated by a so-called solid phase method (triester method) using aminated CPG as a carrier.
Description
SPECIFICATION
A method of synthesizing long chain DNA
The present invention relates to a novel method of synthesizing long chain DNA carrying information for synthesis of specific proteins and, more particularly, it relates to a method of synthesizing long chain DNA purely chemically, i.e. without the use of enzymes.
It has been known that the synthesis of polypeptides by a gene technological means using synthetic gene is possible by steps of (1 ) synthesis of structural gene; (2) recombination of the gene into suitable plasmid; (3) transformation of suitable host by the formed chimera plasmid; and (4) obtaining of the desired polypeptide by culturing the transformed substance.
Recently, developments of DNA probe is attracting public attention as a novel means for gene technology.
This is a a method of identifying unknown DNA and RNA which is a transcribed product by a hybridization of single stranded DNA and RNA which are known in the art by utilizing properties of DNA and RNAthat they form duplex by selecting complimentary substance just like the relation of template and casting. Since very sensitive and prompt identification is possible by utilizing the hybridization method, said method can be applied for diagnosis of precise name of disease by finding specific DNA and RNA in a gene level from blood and cells of patients and pathogenic bacteria. Accordingly, DNA has important value as diagnostic agent by its utilization as DNA probe.
With reference to the above-given DNA as structural gene and that as a source of utilization as DNA probe, it has been known in terms of its nature that the longer the base sequence, the more important as information source and the wider in utilization range to DNA probe. However it has been also known that the longer the base sequence, the more difficult in its synthesis.
Consequently, development of technique in synthesizing long chain DNA by easy manner has been desired.
Conventional method for synthesizing DNA is as follows. Thus, first, comparatively short DNA fragment with 10 to 20 basic residues is chemically synthesized, then they are combined to prepare fragments having total structure of double stranded DNA exhibiting information on desired peptide synthesis, and finally they are combined using an enzyme called DNA ligase.
However, by such a method, only comparatively short fragments with 1 (monomer), 2 (dimer) or 3 (trimer) bases are manufactured prior to block condensation and it is not possible to synthesize long DNA with 80 residues or the like.
In addition, in said method, it is essential to use an enzyme called DNA ligase. Therefore, in synthesizing double stranded DNA as gene, it is necessary that all base sequences constituting double stranded DNA are synthesized at one time. Accordingly, the above method is not so effective in a process of synthesizing double stranded DNA.
The present inventors have continued studies in order to overcome the above technical difficulty and have succeeded in synthesizing DNA with 46 bases or so by utilizing a method called a triester method (among the so-called solid methods) in which 1% polystyrene is used as a support and the compounds of 4 (tetramer) or 5 (pentamer) bases are subjected to a repeated condensation.
Even by such a method, however, the base numbers in the resulting DNA are 50 at the largest and there is still a difficulty in synthesizing DNA with chains of as long as 80 to 150 residues.
(Problems that the Present Invention Solves)
In view of the above, the present inventors have further carried out continued studies paying their attention to (1 ) the synthesis of long chain DNA carrying as much as gene information and (2) the synthesis under more advantageous conditions and finally achieved the present invention.
According to the present invention there is provided:
A method of synthesizing long chain DNA, characterised in that, blocks having 4 to 8 base sequences are purely chemically ligated by a so-called solid phase method (triester method) using aminated controlled pore glass as a carrier.
The present invention will be further illustrated as hereunder:
Each block prior to the condensation can be obtained by the conventional way in which each base is subjected to a liquid phase synthesis.
Aminated CPG (controlled pore glass) (cf. Tetrahedron,24, 747-750, 1983) used in the present invention is used as a carrier in the solid phase method. To the amino group of this substance is combined deoxythymidine which is changed to 3'-sucinate by usual method. This is used as a carrier for nucleoside.
Each desired block is extended, on this resin, to the direction of 5'-terminal successively. As to condensation agent, mesitylene sulfonyl-3-nitrotriazolide (MSNT) can be used, for example. The hereby resulting DNA is single stranded and the complimentary strand DNA which is necessary for preparation of duplet DND can be easily obtained by the similar way. Or such duplet DNA can be very easily obtained by the use of DND polymerase using short fragment (10 b.p. or so) which is complimentary with 3"-terminal region of the resulting single stranded DNA. The fact that DNA polymerase can be used is one of the most advantageous merits of the present invention that the condensation reaction can be accomplished without the aid of DNA ligase which has been widely used in conventional methods.
The resulting duplet DNA is combined to give vector plasmid by the known method, then transformed to bacteria such as Escherichia coli, and the strain is cultured to afford desired polypeptide. In the above steps, various gene technological means which have been already established can be applied.
It is possible in accordance with the present invention to synthesize DNA with as long as 80 to 150 residues and, therefore, polypeptides with 15 to 30 amino acids can be synthesized by the known gene technological means. For instance, the following polypeptides can be synthesized. They are growth hormone-release inhibiting factor (Somatostatin, containing 14 amino acids), stomach acid secreting stimulant (Gastrin, containing 17 amino acids), duodenum ulcer remedy (Secretin, containing 27 amino acids), stimulant for secretion of growth hormone, insuline and blood sugar level increase (Glucagon, containing 29 amino acids), morphine like agent (beta-Endorphin, containing 31 amino acids), and hypercalcemia remedy (Calcitonin, containing 32 amino acids), and the like.
In addition, the long chain DNA of the present invention is applied not only for DNA base sequences of structural gene parts but also for the manufacture of general DNA including regulatory sites and specific sequences as well as for long chain DNA probe recognizing their structures. Accordingly the present invention can be positively applied for development of diagnostic agents.
(Effect the Invention) According to the present invention, long chain DNA can be synthesized simply and in large quantities. The long chain DNA of the present invention can be effectively utilized as (1) gene information source concerning polypeptide synthesis and (2) a source for application of DNA probe in view of gene technology.
Production of DNA has been 0.1 OD (1 OD is equivalent to about 50 micrograms) per one lot at best.
However, in accordance with the present invention, it is now possible to manufacture in quantities as large as 30 to 50 OD per lot. Consequently, expansion of utilizable field of long chain DNA as a gene and as a DNA probe can be expected.
(Examples)
The present invention is further illustrated by giving examples concerning synthesis of endorphin whose physiological activities such as central nervous analgesic action and endocrine hormone action have been known.
(1 ) Synthesis of each block constituting base sequences including endorphin gene.
Amino acid sequence of endorphins has been known and the DNA base sequence corresponding thereto can be freely selected by referring to a table of coden usage. They are given as hereunder together with their relation between each block constituting DNA base sequences used in the present invention. The upper, middle and lower columns are each block (figures therein are block numbers), base sequence and corresponding amino acid sequence, respectively. Incidentally, restricted enzyme sites are given at both terminals of DNA base sequences. Said sites are used in inserting plasmid.
Gi - Endorphin
13 9 12 s 10 5' ACCTGCAGCCCGT CGC TAC GGT GGTTTC ATG Pst I Arg Arg Tyr Gly Gly Phe Met v 9 > 8 ^ 7 jt 6 s ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG Thr Ser Glu Lys Ser Gln Thr Pro Leu Val 2 2 1 5-c4 - 3 - ACT TAA TAG GGCTGCAGGT 3' Thr STOP STOP Pstl # α;- [Leu5] - Endorphin
131615 109 5' ACCTGCAGCC ATG TAC GGT GGT TTC TTG Pst I Met Tyr Gly Gly Phe Leu e-- 9 8 6 ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG Thr Ser Glu Lys Ser Gln Thr Pro Leu Val 2 5 - 4 3 ACT TAA TAG GGCTGCAGGT I 3' Thr STOP STOP Pstl # #- [Leu5 ]- Endorphin
- 13 16 15 5 IACCTGCAGCC ATG TAC GGTGGTTTC TTG Pst I Met Tyr Gly Gly Phe Leu 9 9 8 ' 7 6 ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG Thr Ser Glu Lys Ser Gln Thr Pro Leu Val 2 1 5 at 17 ;;' 3 ACT TTG TAG GGCTGCAGGT13' Thr Leu STOP Pst I # #-endorphin
13 ) 12 x 11 10 5' 1 ACCTGCAGCC CGT CGC TAC GGT GGT TTC ATG Pst I Arg Arg Tyr Gly Gly Phe Met < -9 9 & --------xxx---7--"- 6 --e ACT TCT GAG AAG TCT CAA ACT CCA TTG GTG Thr Ser Glu Lys Ser Gln Thr Pro Leu Val 2 1 5 17 8^ 3 sc x ACT TTG TAG GGCTGCAGGT, 3' Thr Leu STOP Pst I Among the blocks constituting the above endorphin genes, the block 7 was synthesized by the steps as given below.
bzbz (I) d (DnTr) AeAecE TEA bzbz (II) d d (OnTr) Aece CE AECECE bzbz BSA bz (m) d (DnTr) AeAeo- d (DHTr) TeCecE (V) . . (IV) bzbz TEA bzbz dA(E Ce CE d (DnTr) Ce Ae CE llSNT bz bz BSA d (DnTr) TeCeo bzbzbzbz d (is) ta) , d (DnTr) Ae Ae Ae Ce CE bzbz |TEl dCe Ae CE TEA 1 ( . ) M SìlT bzbzbzbz å (DMIr) AereAeceo- bzbzbz (X I) d (DnTr) TececeAecE 1 BSA cx, bzbzbz dT2C2 2 Ae CE . I (X E) nsT CX 11) bzbzbzbi bzbbi d (D:ITr) C.ep.eC2TeCeCe.4ecE I lba (XIY) bzbzlzSz bbzbz d (DT) PeAePeceTecee':eo- ----Block 7 DMTr: 4,4'-Dimethoxytolityl bz A: N-Benzoyladenosyl bz C: N-Benzoylcytidyly1
T: Tymidylyl #: o-Chlorophenyl phosphate BSA: Benzenesulfonic acid
TEA:Triethylamine
Other blocks (1-6, and 8-17) constituting endorphin type genes can be synthesized by similar way. Each yield is given as hereunder.
Blocks Base Sequences Yield
1 CAGG 77
2 GCTG 94
3 TAGG 70
4 TTAA 104
5 TGAC 93
6 TTGG 67
7 AAACTCCA 70
8 GAAGTCTC 62
9 ACTTCTGA 65
10 GTTTCATG 65
11 CTACGGTG 70
12 GCCCGTCA 65
13 ACCTGCA 65
14 GTTTCTTG 70
15 GTACGGTG 67
16 GCCAT 75
17 TTTG 95 (2) Endorphins genes synthesis:
alpha-Endorphin gene (deoxy 80 mer) containing restricted enzyme sites was synthesized by a solid phase method as follows.
1. Deoxytymidine CPG resin is washed with CH2CI2/MeOH.
2. Detritylation is conducted with 2% BSA/CH2CI2 (this was conducted repeatedly and promptly until
colorization disappears)
3. Subjected to azeotropic drying after substituted with pyridine.
A solution of each block is added, subjected to azeotropic drying, and MSNT and pyridine for the
reaction are added. Allowed to stand at room temperature and washed with pyridine.
4. 0.1 M Dimethylaminopyridine/pyridine solution and acetic anhydride are added, allowed to stand at
room temperature, and washed with pyridine.
The above operation is conducted repeatedly, for 13 times in total. Average yield of this reaction was 84%.
Then the resin is deprotected, at room temperature, with a solution of 0.1 M tetramethylguanidine-pyridine aldoxime (cf. C.B. Reese, et al: Tetrahedron Lett., 2727, 1978) in dioxane-water, then washed with pyridine-water, the washing is concentrated in vacuo, concentrated ammonia water is added thereto, and the mixture is warmed. Ammonia is evaporated therefrom and a part of the residue is taken using dimethyoxytrityl group as a target to calculate the yield of the final stage.
The residual reaction solution is subjected to a reversed phase (C18 silica gel for Prep 500 manufactured by
Waters), ion exchange (DEAE-toyopal), and reversed phase (C18 silica gel, TSK-Gel 10-20 micrometers) open chromatographies to afford pure alpha-endorphin gene (containing restricted enzyme sites) (- deoxy 80 mer).
Purity was confirmed by HPLC (Nucleosil 300-7 C18) and by electrophoresis and its base sequences were confirmed by Maxam-Gilbert method. The result is given in Figure 1 to Figure 3.
Similarly prepared were alpha-(Leu5)-endorphin gene (containing restrictive enzyme site) (deoxy 77 mer), gamma-(Leu5)-endorphin gene (containing restrictive enzyme site) deoxy 77 mer) and gamma-endorphin gene (containing restrictive enzyme site) (deoxy 80 mer).
(3) Synthesis ofdupletDNA and its combination with vectorplasmid.
Each one mole of deoxy 80 mer and synthetic nucleotide primer which is complimentary with 3'-terminal of the former were mixed, heated at 65"C, and cooled to room temperature to anneal the deoxy 80 mer and the primer. Then E. coli polymerase I (Klenow fragment) was added by conventional may and made to react at 37"C for 30 minutes so that DNA was made into double stranded.
DNA was recovered as a precipitate in ethanol, made to react at 27 C for 30 minutes using T4 polynucleotidekinase, and both 5'-terminals of the double stranded DNA were phosphorylated.
Then the vector plasmid pUC 8 DNA was scissored with a restrictive enzyme Pst 1, added to the above double stranded DNA solution, made to react at 16"C overnight with T4 DNA ligase, and the double stranded d 80 mer DNA was combined with the vector plasmid.
(4) Cloning ofplasmid containing endorphin gene.
The plasmid prepared as above was transformed into E. coli JM 103 strain by conventional way, then selected using a deficiency of beta-galactosidase activity present in the pUC 8 as a target, and plasmid molecules were collected by cloning from the strain.
It has been confirmed that plasmid in which endorphin gene was inserted into the correct orientation and position as desired in accordance with Maxam-Gilbert method.
(5) Obtaining ofendorphins.
Transformed E. coliJM 103 strain was precultured overnight in an LB medium, planted in 2YT medium, and subjected to a shake culture at 370C.
IPTG was added to the logarithmic productive phase stages (initial, medium and final stages) to make it 0.5mM and synthesis of endorphin was induced. After being induced by IPTG, fused protein was extracted, and analyzed by HPLC whereupon it was found that adequate quantity of protein production was observed (1-5.0 x 105 molecules per cell) when induction was applied at the initial stage of logarithmic productive phase.
With reference to natural type alpha-endorphin and gamma-endorphin hacing methionine residue in a molecule, they were treated with trypsin by conventional way. With reference to alpha-(Leu5)-endorphin and gamma-(Leu5)-endorphin having leucine residue in place of methionine, they were treated with BrCN.
Anyway, each of desired endorphin proteins desired was subjected to a column chromatography according to the general purification method of proteins whereupon each of them was separated and purified.
The fact that each of the resulting endorphin molecules exhibits desired amino acid sequence was confirmed by the fact that they were identical with the samples already obtained by the peptide synthesis by testing with HPLC using a reverse phase carrier.
4. Brief Explanation ofDrawings:
Figure 1 is X-ray autoradiogram showing the result of 20% polyacryamide electrophoresis of deoxy 80 mer containing alpha-endorphin gene synthesized after determination by Maxam-Gilbert method.
Figure 2 is X-ray autoradiogram showing the result of 8% polyacrylamido electrophoresis of deoxy 80 mer containing alpha-endorphin gene synthesized after determination by Maxam-Gilbert method.
Figure 3 shows the result of high performance liquid chromatography (Nucleosil 300-7 C18) of deoxy 80 mer containing alpah-endorphin gene synthesized after determination by Maxam-Gilbert method. Ordinate and abscissa show absorbancy and time, respectively. Solvent system used was triethylamine acetateacetonitrile and the flowing speed was 1.0 ml/min.
Claims (3)
1. A method of synthesizing long chain DNA, characterised in that, blocks having 4 to 8 base sequences are purely chemically ligated by a so-called solid phase method (triester method) using aminated controlled pore glass as a carrier.
2. A method as claimed in Claim 1 in which the blocks having 4 to 8 base sequences are subject to condensation reaction.
3. A method of synthesizing long chain DNA and which is substantially as described herein.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59281645A JPS61152695A (en) | 1984-12-26 | 1984-12-26 | Synthesis of long-chain dna |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8530915D0 GB8530915D0 (en) | 1986-01-29 |
| GB2169605A true GB2169605A (en) | 1986-07-16 |
| GB2169605B GB2169605B (en) | 1989-06-07 |
Family
ID=17641986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8530915A Expired GB2169605B (en) | 1984-12-26 | 1985-12-16 | A method of synthesizing long chain dna |
Country Status (9)
| Country | Link |
|---|---|
| JP (1) | JPS61152695A (en) |
| KR (1) | KR940000543B1 (en) |
| CA (1) | CA1295558C (en) |
| CH (1) | CH672791A5 (en) |
| DE (1) | DE3544459C2 (en) |
| ES (1) | ES8802330A1 (en) |
| FR (1) | FR2575162B1 (en) |
| GB (1) | GB2169605B (en) |
| IT (1) | IT1208725B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5114839A (en) * | 1988-05-24 | 1992-05-19 | Gesellschaft Fur Biotechnologische Forsching Mbh | Process for dna sequencing using oligonucleotide bank |
| WO1993021203A1 (en) * | 1992-04-15 | 1993-10-28 | The Johns Hopkins University | Synthesis of diverse and useful collections of oligonucleotides |
| WO1994014972A1 (en) * | 1992-12-23 | 1994-07-07 | Edward David Hyman | Method and apparatus for enzymatic synthesis of oligonucleotides |
| WO2001088173A3 (en) * | 2000-05-16 | 2002-06-06 | Hercules Inc | Methods for the enzymatic assembly of polynucleotides and identification of polynucleotides having desired characteristics |
| US7205399B1 (en) | 2001-07-06 | 2007-04-17 | Sirna Therapeutics, Inc. | Methods and reagents for oligonucleotide synthesis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2007670A (en) * | 1977-11-08 | 1979-05-23 | Genentech Inc | Triester process for the synthesis of oligonucleotides |
| EP0035719A2 (en) * | 1980-02-29 | 1981-09-16 | University Patents, Inc. | Process for producing modified inorganic polymers, their use in producing polynucleotides, and a reagent useful in these processes |
| EP0090789A1 (en) * | 1982-03-26 | 1983-10-05 | Monsanto Company | Chemical DNA synthesis |
| GB2125798A (en) * | 1982-08-20 | 1984-03-14 | Genex Corp | Solid phase synthesis of oligonucleotides |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0102995A1 (en) * | 1982-03-08 | 1984-03-21 | Celltech Limited | Polynucleotide synthesis |
| DE3301833A1 (en) * | 1983-01-20 | 1984-07-26 | Gesellschaft für Biotechnologische Forschung mbH (GBF), 3300 Braunschweig | METHOD FOR SIMULTANEOUS SYNTHESIS OF SEVERAL OLIGONOCLEOTIDES IN A SOLID PHASE |
| AU575586B2 (en) * | 1983-09-02 | 1988-08-04 | Syngene, Inc. | Oligonucleotide synthesis employing primer with oxidzable substituents in system |
| GR80965B (en) * | 1983-11-21 | 1985-03-20 | Ciba Geigy Ag | Method for the preparation of protease inhibitors |
-
1984
- 1984-12-26 JP JP59281645A patent/JPS61152695A/en active Granted
-
1985
- 1985-12-16 GB GB8530915A patent/GB2169605B/en not_active Expired
- 1985-12-16 DE DE3544459A patent/DE3544459C2/en not_active Expired - Fee Related
- 1985-12-18 IT IT8548954A patent/IT1208725B/en active
- 1985-12-20 FR FR858518965A patent/FR2575162B1/en not_active Expired
- 1985-12-20 CH CH5457/85A patent/CH672791A5/fr not_active IP Right Cessation
- 1985-12-23 CA CA000498535A patent/CA1295558C/en not_active Expired - Lifetime
- 1985-12-24 ES ES550390A patent/ES8802330A1/en not_active Expired
- 1985-12-26 KR KR1019850009935A patent/KR940000543B1/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2007670A (en) * | 1977-11-08 | 1979-05-23 | Genentech Inc | Triester process for the synthesis of oligonucleotides |
| EP0035719A2 (en) * | 1980-02-29 | 1981-09-16 | University Patents, Inc. | Process for producing modified inorganic polymers, their use in producing polynucleotides, and a reagent useful in these processes |
| EP0090789A1 (en) * | 1982-03-26 | 1983-10-05 | Monsanto Company | Chemical DNA synthesis |
| GB2125798A (en) * | 1982-08-20 | 1984-03-14 | Genex Corp | Solid phase synthesis of oligonucleotides |
Non-Patent Citations (2)
| Title |
|---|
| WO A1 83/03098 * |
| WO A1 85/01051 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5114839A (en) * | 1988-05-24 | 1992-05-19 | Gesellschaft Fur Biotechnologische Forsching Mbh | Process for dna sequencing using oligonucleotide bank |
| WO1993021203A1 (en) * | 1992-04-15 | 1993-10-28 | The Johns Hopkins University | Synthesis of diverse and useful collections of oligonucleotides |
| WO1994014972A1 (en) * | 1992-12-23 | 1994-07-07 | Edward David Hyman | Method and apparatus for enzymatic synthesis of oligonucleotides |
| US5602000A (en) * | 1992-12-23 | 1997-02-11 | Hyman; Edward D. | Method for enzymatic synthesis of oligonucleotides |
| US5629177A (en) * | 1992-12-23 | 1997-05-13 | Hyman; Edward D. | Method for the enzymatic synthesis of oligonucleotides using thermostable 3'-phosphatase |
| WO2001088173A3 (en) * | 2000-05-16 | 2002-06-06 | Hercules Inc | Methods for the enzymatic assembly of polynucleotides and identification of polynucleotides having desired characteristics |
| US6479262B1 (en) | 2000-05-16 | 2002-11-12 | Hercules, Incorporated | Solid phase enzymatic assembly of polynucleotides |
| US6635453B2 (en) | 2000-05-16 | 2003-10-21 | Hercules Incorporated | Methods for the enzymatic assembly of polynucleotides and identification of polynucleotides having desired characteristics |
| US7205399B1 (en) | 2001-07-06 | 2007-04-17 | Sirna Therapeutics, Inc. | Methods and reagents for oligonucleotide synthesis |
Also Published As
| Publication number | Publication date |
|---|---|
| GB8530915D0 (en) | 1986-01-29 |
| ES550390A0 (en) | 1988-05-01 |
| FR2575162B1 (en) | 1989-05-05 |
| IT8548954A0 (en) | 1985-12-18 |
| JPH0586400B2 (en) | 1993-12-10 |
| GB2169605B (en) | 1989-06-07 |
| KR940000543B1 (en) | 1994-01-24 |
| CA1295558C (en) | 1992-02-11 |
| ES8802330A1 (en) | 1988-05-01 |
| CH672791A5 (en) | 1989-12-29 |
| DE3544459C2 (en) | 1993-12-16 |
| KR860005025A (en) | 1986-07-16 |
| JPS61152695A (en) | 1986-07-11 |
| DE3544459A1 (en) | 1986-07-03 |
| FR2575162A1 (en) | 1986-06-27 |
| IT1208725B (en) | 1989-07-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR0139629B1 (en) | Mini-proinsulin, method and use of the preparation thereof | |
| JPH0657154B2 (en) | Cloning vehicle for expressing desired polypeptide | |
| IL74626A (en) | Insulin-like growth factor i | |
| EP0119621A1 (en) | Interleukin-2 | |
| US5149657A (en) | Escherichia coli expression vector encoding bioadhesive precursor protein analogs comprising three to twenty repeats of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) | |
| NL192116C (en) | Beta-urogastron genes, corresponding recombinant plasmids, corresponding transformants and their preparation, and of beta-urogastron. | |
| WO1983004030A1 (en) | The manufacture and expression of genes for urogastrone and polypeptide analogs thereof | |
| EP0108387B1 (en) | Preparation of recombinant growth releasing factors | |
| CA1295558C (en) | Synthesis of long chain dna | |
| US4842996A (en) | Probes comprising modified adenine groups, their preparation and their uses | |
| NO844123L (en) | PROCEDURE FOR THE PREPARATION OF NONAPEPTIDES AND DEATH COPETIDES | |
| EP0095351B1 (en) | A precursor of a c-terminal amidated peptide and production thereof | |
| US4728609A (en) | Recombinant growth hormone releasing factor | |
| WO1983004028A1 (en) | The manufacture and expression of genes for calcitonin and polypeptide analogs thereof | |
| US5474913A (en) | Process for the preparation of motilin-like polypeptide and expression thereof | |
| SU1727533A3 (en) | Method for preparation of water-soluble component of human receptor with low affinity f @@@ | |
| US5252482A (en) | Precursor of a C-terminal amidated calcitonin | |
| JP2575022B2 (en) | Human motilin precursor, cloned DNA containing the precursor, method for preparing the same, and plasmid incorporating the cloned DNA | |
| Rocchi et al. | Synthesis of peptide analogs of the N-terminal eicosapeptide sequence of ribonuclease A. XIII. Synthesis of des-Lys7-[Orn10]-and des-Phe8-[Orn10]-S-peptides | |
| Robins et al. | Nucleoside peptides. I. Synthesis of 5'-deoxy-5'amino-5'-N-aminoacyl peptide derivatives of guanosine, adenosine, and 2'-deoxyadenosine and their effect of cell-free protein synthesis | |
| EP0622459A1 (en) | Fused proteins for preparing vasoactive intestinal polypeptide analogs, method of preparing same and recombinant plasmids and transformant microorganisms | |
| JP2561060B2 (en) | Improved RNase T1 | |
| US20030157505A1 (en) | Novel substance library and supramolecular complexes prepared therewith | |
| JP2793216B2 (en) | Sorbin and derived peptides to enhance mucosal absorption | |
| GB2164650A (en) | Process for production of y-interferon |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19941216 |