NO161572B - Fremgangsmaate for spalting av dobbelttraadet dna. - Google Patents
Fremgangsmaate for spalting av dobbelttraadet dna. Download PDFInfo
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- NO161572B NO161572B NO84843718A NO843718A NO161572B NO 161572 B NO161572 B NO 161572B NO 84843718 A NO84843718 A NO 84843718A NO 843718 A NO843718 A NO 843718A NO 161572 B NO161572 B NO 161572B
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- dna
- stranded
- primer
- stranded dna
- polymerase
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- 238000000034 method Methods 0.000 title claims description 15
- 108020004414 DNA Proteins 0.000 claims description 19
- 238000003776 cleavage reaction Methods 0.000 claims description 16
- 230000007017 scission Effects 0.000 claims description 16
- 102000053602 DNA Human genes 0.000 claims description 15
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 4
- 230000001036 exonucleolytic effect Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 229940104302 cytosine Drugs 0.000 claims description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 2
- 239000001226 triphosphate Substances 0.000 claims description 2
- 235000011178 triphosphate Nutrition 0.000 claims description 2
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 2
- 239000013615 primer Substances 0.000 description 10
- 239000012634 fragment Substances 0.000 description 9
- 108060002716 Exonuclease Proteins 0.000 description 5
- 102000013165 exonuclease Human genes 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
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- 108091008146 restriction endonucleases Proteins 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 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
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 230000006820 DNA synthesis Effects 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
- 239000004471 Glycine Substances 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010046075 Thymosin Proteins 0.000 description 1
- 102000007501 Thymosin Human genes 0.000 description 1
- 238000000211 autoradiogram Methods 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
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- 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
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- 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
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
-
- 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
- C07K14/575—Hormones
- C07K14/655—Somatostatins
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- 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
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Description
Foreliggende oppfinnelse vedrører en fremgangsmåte for spalting av dobbelttrådet DNA på et hvilket som helst gitt punkt.
Rekombinante DNA-metoder innebærer ofte spalting av dobbelttrådet DNA på en kontrollert måte, men restriksjonsenzymer spalter bare DNA'en ved gjenkjennelsesseter (dvs. nukleotid-sekvenser) som er spesifikke for det angjeldende enzym. Foreliggende oppfinnelse tilveiebringer en metode for spalting av dobbelttrådet DNA ved et hvilket som helst ønsket punkt, selv i fravær av et restriksjonsenzymsete, en teknikk som bl.a. er nyttig ved dannelsen av trp-operoner som har attenuator-delesjoner andre enn dem som tidligere er oppnådd ved seleksjon av mutanter.
Ifølge foreliggende oppfinnelse er det således tilveiebragt en fremgangsmåte for spalting av dobbelttrådet DNA på et hvilket som helst gitt punkt, og denne fremgangsmåten er kjennetegnet ved at man: (a) omdanner den dobbelttrådede DNA til enkelttrådet DNA i et område som omgir det tilsiktede spaltningspunkt; (b) hybridiserer til det enkelttrådede området som dannet i trinn (a) en komplementær primer av den enkelttrådede DNA, hvor 5'-enden til primeren ligger mot-satt det nukleotid som anslutter seg til det tilsiktede spaltningsprodukt; (c) restituerer den del av den andre tråden som ble eliminert i trinn (a), som ligger i 3'-retningen fra nevnte primer, ved en reaksjon med DNA-polymerase i nærvær av adenin-, thymin-, og cytosinholdige deoksynukleotidtrifosfåter; og (d) nedbryter den gjenværende enkelttrådede lengde av DNA
som stikker ut forbi det tilsiktede spaltningspunkt.
Foreliggende oppfinnelse vil bli mer spesielt illustrert i det nedenstående under henvisning til den medfølgende tegning som illustrerer foreliggende fremgangsmåte.
Generelt blir dobbelttrådet DNA omdannet til enkelttrådet DNA I området som omgir det tilsiktede spaltningspunkt, som ved reaksjon med lambda-eksonuklease. En syntetisk eller annen enkelttrådet DNA-primer hybridiseres deretter til den tidligere dannede enkelttrådede lengden, ved Watson-Crick-basepar-dannelse, idet primersekvensen er slik at det sørges for at dens 5'-ende vil bli koterminal med nukleotidet på den første tråden like før det tilsiktede spaltningspunktet. Primeren blir deretter forlenget i 3'-retningen ved reaksjon med DNA-polymerase, hvorved den delen av den dobbelttrådede DNA før den tilsiktede spaltning som gikk tapt i det første trinnet, gjenskapes. Samtidig eller deretter blir den del av den første tråden som befinner seg utenfor det tilsiktede spaltningspunktet, fjernet ved nedbrytning. Dette kan illustreres som angitt nedenfor, hvor "v" markerer det tilsiktede spaltningspunkt:
v
a) ..... tilsiktet spaltningspunkt "v"
v
b) ..... gjort enkelttrådet omkring "v"
v
c) ..... primer-hybridisering
. • • • • $-/\^\^\A^
V
d) ..... forlengelse fra primer
e)
enkelttråd-nedbrytning
I den mest foretrukne utførelsen blir trinnene (d) og (e) foretatt samtidig ved anvendelse av en polymerase som samtidig nedbryter den fremstikkende enkelttrådede enden i 3'-» 5' retningen og forlenger primeren (i nærvær av dATP, dGTP, dTTP og dCTP) i 5' -» 3' retningen. Materialet som fore-trekkes for dette formål, er Klenow polymerase I, dvs. det fragment som oppnås ved proteolytisk spalting av DNA polymerase I som inneholder den 5' -> 3' polymeriserende aktivitet og den 3' -» 5' eksonukleolytiske aktivitet fra stamenzymet, og likevel mangler dets 5' -» 3' eksonukleolytiske aktivitet (A. Kornberg, DNA Synthesis, 98, W.H. Freeman and Co., SFO
(1974)).
Ved bruk av samme fremgangsmåte som beskrevet ovenfor kan attenuator-delesjoner foretas på en hvilken som helst ønsket måte i et trp-operonholdig plasmid som først er linearisert ved f.eks. spalting ved et restriksjonssete nedstrøms fra det punkt ved hvilket molekylet skal gjøres buttendet ("v" ovenfor". Resirkulariserlng etter delesjon av attenuatorområdet kan bevirkes f.eks. ved buttende-ligering eller på annen måte som vil være nærliggende for en fagmann. Foreliggende oppfinnelse anvendes i metoder som er beskrevet i NO patent-søknad nr. 810986 og i NO patentsøknad nr. 843719.
Under henvisning til tegningen ble plasmid pSom7A2 (se NO patentsøknad nr. 810986 og nr. 843719) Hind III nedbrutt, fulgt av nedbrytning med lambda-eksonuklease (en 5' til 3' eksonuklease) under betingelser valgt slik at det ble oppnådd nedbrytning utenfor Bgl Il-restriksjonssetet i det LE'-kodende området. 20 pg av Hind III-nedbrutt pSom7A2 ble oppløst i buffer (20 mM glycinbuffer, pH 9,6, 1 mM MgCl2, 1 mM e-merkaptoetanol). Den resulterende blanding ble behandlet med fem enheter av lambda-eksonuklease i 60 min. ved romtemperatur. Den oppnådde reaksjonsblanding ble deretter f enolekstrahert, kloroformekstrahert og etanolutfelt.
For til slutt å danne en EcoRI-rest ved distalenden av LE'-genfragmentet ble en primer <32>pCCTGTGCATGAT syntetisert ved hjelp av den forbedrede fosfotriestermetoden (R. Crea et al., Proe. Nat'l. Acad. Sei. USA 75, 5765 [1978]) og hybridisert til den enkelttrådede enden av LE'-genfragmentet som resul-terte fra lambda-eksonukleasenedbrytning. Hybridiseringen ble foretatt som beskrevet i det nedenstående. 20 pg av det lambda-eksonukleasebehandlede Hind III-ned-brytningsproduktet av plasmid pSom7A2 ble oppløst i 20 pl H2O og kombinert med 6 pl av en oppløsning inneholdende ca. 80 picomol av det 5'-fosforylerte oligonukleotidet som beskrevet ovenfor. Det syntetiske fragmentet ble hybridisert til 3' t-enden av den LE'-kodende sekvens, og den gjenværende enkelttrådede delen av LE'-fragmentet ble fylt inn ved hjelp av Klenow-polymerase I-metoden beskrevet ovenfor, ved bruk av dATP, dTTP, dGTP og dCTP.
Reaksjonsblandingen ble oppvarmet til 50°C og fikk langsomt avkjøles til 10°C, hvoretter 4 pl Klenow-enzym ble tilsatt. Etter 15 min.s inkubasjon ved romtemperatur fulgt av 30 min.s inkubasjon ved 37°C, ble reaksjonen stoppet ved tilsetning av 5 pl av 0,25 molar EDTA. Reaksjonsblandingen ble fenol-ekstrahert, kloroformekstrahert og etanolutfelt. DNA'en ble deretter spaltet med restriksjonsenzymet Bgl II. Fragmentene ble separert ved hjelp av PAGE. Et autoradlogram oppnådd fra gelen viste et 32p_meri{et fragment av den forventede lengde på ca. 470 bp som ble utvunnet ved elektroeluering. Som angitt har dette fragmentet LE' (d) et Bgl II-sete og en butt ende som sammenfaller med begynnelsen av primeren.
Dette kan deretter f.eks. benyttes som beskrevet i NO patent-søknad nr. 810986og nr. 843719 til å danne en ekspres-jonsvektor inneholdende LE' (d )-fragmentet (29) under styring av en promotor (trp promotor-operator), i hvilken vektor man på funksjonell måte kan innføre den kodende sekvensen for et heterologt polypeptid slik som thymosin alfa- en, human proinsulin, eller A- eller B-kjeden i human insulin. Den resulterende ekspresjonsvektoren kan deretter transformeres 1 en egnet vert (E. coli stamme 294 ) for å uttrykke det heterologe peptidet.
Claims (3)
1. Fremgangsmåte for spalting av dobbelttrådet DNA på et hvilket som helst gitt punkt, karakterisert ved at man: (a) omdanner den dobbelttrådede DNA til enkelttrådet DNA i et område som omgir det tilsiktede spaltningpunkt; (b) hybridiserer til det enkelttrådede området som dannet i trinn (a) en komplementær primer av den enkelttrådede DNA, hvor 5'-enden til primeren ligger mot-satt det nukleotid som anslutter seg til det tilsiktede spaltningspunkt; (c) restituerer den del av den andre tråden som ble eliminert i trinn (a), som ligger i 3'-retningen fra nevnte primer, ved en reaksjon med DNA-polymerase i nærvær av adenin-, thymin-, guanin- og cytocinholdige deoksynukleotidtrifosfåter; og (d) nedbryter den gjenværende enkelttrådede lengde av DNA som stikker ut forbi det tilsiktede spaltningspunkt.
2. Fremgangsmåte ifølge krav 1, karakterisert ved at trinne (c) og (d) utføres samtidig ved en reaksjon av det i trinn (d) dannede produkt med DNA-polymerase som polymeriserer i retningen 5' -» 3', er eksonukleolytisk i retningen 3' -» 5', men ikke eksonukleolytisk I retningen 5 ' -+ 3 ' .
3. Fremgangsmåte ifølge krav 2, karakterisert ved at nevnte polymerase er Klenow polymerase
Priority Applications (1)
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NO84843718A NO161572C (no) | 1980-03-24 | 1984-09-18 | Fremgangsmaate for spalting av dobbelttraadet dna. |
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US13329680A | 1980-03-24 | 1980-03-24 | |
NO810986A NO810986L (no) | 1980-03-24 | 1981-03-23 | Bakteriell polypeptid-ekspresjon under anvendelse av tryptofon-promoter-operator |
NO84843718A NO161572C (no) | 1980-03-24 | 1984-09-18 | Fremgangsmaate for spalting av dobbelttraadet dna. |
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NO843718L NO843718L (no) | 1981-09-25 |
NO161572B true NO161572B (no) | 1989-05-22 |
NO161572C NO161572C (no) | 1989-08-30 |
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NO810986A NO810986L (no) | 1980-03-24 | 1981-03-23 | Bakteriell polypeptid-ekspresjon under anvendelse av tryptofon-promoter-operator |
NO84843719A NO165644C (no) | 1980-03-24 | 1984-09-18 | Fremgangsmaate for dannelse av et ekspresjonsplasmid for ekspresjon av et heterologt gen. |
NO84843718A NO161572C (no) | 1980-03-24 | 1984-09-18 | Fremgangsmaate for spalting av dobbelttraadet dna. |
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NO810986A NO810986L (no) | 1980-03-24 | 1981-03-23 | Bakteriell polypeptid-ekspresjon under anvendelse av tryptofon-promoter-operator |
NO84843719A NO165644C (no) | 1980-03-24 | 1984-09-18 | Fremgangsmaate for dannelse av et ekspresjonsplasmid for ekspresjon av et heterologt gen. |
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