DK1915446T3 - In vitro rekombination fremgangsmåde - Google Patents

In vitro rekombination fremgangsmåde Download PDF

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DK1915446T3
DK1915446T3 DK06813388.3T DK06813388T DK1915446T3 DK 1915446 T3 DK1915446 T3 DK 1915446T3 DK 06813388 T DK06813388 T DK 06813388T DK 1915446 T3 DK1915446 T3 DK 1915446T3
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dna
molecules
joined
molecule
exonuclease
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Lei Young
Hamilton O Smith
Daniel G Gibson
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Synthetic Genomics Inc
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Claims (17)

1. In vitro fremgangsmåde ved anvendelse af isolerede proteiner til sammenføjning af to dobbeltstrengede (ds) DNA-molekyler af interesse, omfattende: tilvejebringelse af et første dsDNA-molekyle og et andet dsDNA-molekyle der deler en sekvensidentitetsregion ved en ende på hvert DNA-molekyle; og bringe de to DNA-molekyler i kontakt samtidigt med: (a) en oprenset ikke-processiv 5'-exonukiease; (b) et oprenset enkeitstrenget DNA-bindingsprotein (SSB), som accelererer nukleinsyreannealing; (c) en oprenset ikke-strengs-displacerende DNA-polymerase, hvor 5'-exonukleasen og DNA-polymerasen er forskellige enheder; og (d) en oprenset ligase under betingelser hvorved: et 3'-enkeltstrenget overhæng er genereret i hvert molekyle af exonukleasen uden anvendelse af et restriktionenzym; de to enkeitstrengede overhænger annealer for at danne et hullet molekyle; hullerne er udfyldt af polymerasen; og brudene er forsejlet af ligasen, derved sammenføjes molekylerne og danner et intakt dobbeltstrenget DNA-molekyle, hvor en enkelt kopi af sekvensidentitetsregionen er bevaret, hvor ingen af de enzymatiske reaktioner er aktivt afsluttet før begyndelsen af en anden af reaktionerne.
2. Fremgangsmåde ifølge krav 1, hvor exonukleaseaktiviteten er, i det væ- sentlige, Savere end DNA-polymeraseaktiviteten, således at hullerne er udfyldt af polymerasen, i det væsentlige, umiddelbart som de dannes.
3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor (a) den ikke-processive S’-exonuklease er gen 6 produktet fra phag-T7, RedA fra lambda-phag eller RecE fra Rac-prophag; (b) SSB’en er gen 2,5 produktet fra phag-T7, E. coli recA-proteinet, RedB fra lambda-phag eller RecT fra Rac-prophag; (c) DNA-polymerasen er gen 5 produktet fra phag-T7, DNA-polymerasen fra phag-T4 eller E. coli pol I; og/elier (d) ligasen er gen 1,3 produktet fra phag-T7, DNA-ligasen fra phag-T4 eller E. coli-DNA-ligasen.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1-3, hvor et eller flere af DNA-molekylerne der skal sammenføjes er genereret syntetisk.
5. Fremgangsmåde ifølge et hvilket som helst af kravene 1-4, hvor sekvensidentitetsregionen er mindre end 150 basepar i længden, eventuelt omkring 40 bp.
6. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5, yderligere omfattende sammenføjning af ekstra DNA-molekyler til de to DNA-molekyler, hvor for hvert par af ekstra DNA-molekyler, der skal sammenføjes, den distale region af et af DNA-molekylerne i parret deler en sekvensidentitetsregion med den proksimale region af det andet DNA-molekyle i parret.
7. Fremgangsmåde ifølge krav 6, hvor sekvensidentitetsregionen er unik for hvert par af DNA-molekyler.
8. Fremgangsmåde ifølge krav 6 eller krav 7, hvor mindst omkring ti DNA-molekyler er sammenføjet med hinanden i en enkelt reaktionsbehoider.
9. !n vitro-fremgangsmåde til sammenføjning af mere end to dobbeltstrengede DNA-molekyler i en defineret orientering og orden, omfattende (a) udvælgelse af mere end to DNA-molekyler, således at; for hvert par af molekyler der skal sammenføjes, deler molekylerne en sekvensidentitetsregion ved en ende af hvert DNA-molekyle, hvor hver sekvensidentitetsregion er unik for hvert par af DNA-moiekyier der skal sammenføjes; og (b) bringe DNA-molekylerne i kontakt i en reaktionsbianding i en enkelt reaktionsbeholder samtidig med (i) en oprenset ikke-processiv 5'-exonuklease; (ii) et oprenset enkeltstrenget DNA-bindingsprotein (SSB), som accelererer nukleinsyreanneaiing; (iii) en oprenset ikke-strengs-dispiacerende DNA-poiymerase, hvor S'-exonukleasen og DNA-polymerasen er forskellige enheder; og (iv) en oprenset ligase under betingelser hvorved: et 3'-enke!tstrenget overhæng er genereret i hvert molekyle af exonukleasen uden anvendelse af et restriktionsenzym; de enkeltstrengede overhænger annealer for at danne et hullet molekyle; hullerne er udfyldt af polymerasen; og brudene er forsejlet af ligasen derved sammenføjes mere end to dsDNA-molekyler for at danne et intakt duplex-DNA-molekyle hvor sekvensidentitetsregionen er bevaret, hvor ingen af de enzymatiske reaktioner er aktivt afsluttet før begyndelsen af en anden af reaktionerne.
10. Fremgangsmåde ifølge et hvilket som helst af kravene 1-9, hvor de 3'-enkeltstrengede overhæng indeholder sekvensidentitetsregionen.
11. Fremgangsmåde ifølge et hvilket som helst af kravene 1-10, hvor de fire proteiner fungerer på en samordnet måde.
12. Fremgangsmåde ifølge krav 9, hvor et eller flere af de dsDNA-molekyler, der skal sammenføjes er genereret syntetisk.
13. Fremgangsmåde ifølge krav 1 eller krav 12, hvor de dsDNA-molekyler er genereret syntetisk og omfatter tilstødende dele af et gen eller genom af interesse; de dsDNA-molekyler er syntetiseret for at omfatte overlappende sekvensidentitetsregioner ved deres ender; og sammenføjningsreaktionen sammenføjer de dsDNA-molekyler til at danne dele eller hele det syntetiske gen eller genom.
14. Fremgangsmåde ifølge et hvilket som helst af kravene 1-13, hvor sekvensidentitetsregionerne er tilføjet til de dsDNA-molekyier, der skal sammenføjes med PCR-amplifikation.
15. Fremgangsmåde ifølge et hvilket som helst af kravene 1-14, yderligere omfattende udsættelse af de sammenføjede DNA-molekyler for en størrelsesprocedure, isolering af DNA-molekyler med en ønsket længde og indføring af de isolerede DNA-molekyler i en celle af interesse.
16. Fremgangsmåde ifølge et hvilket som helst af kravene 1-4 eller 9-15, hvor mere end de to dsDNA-molekyler omfatter mindst 10 dsDNA-molekyier og for hvert par af DNA-molekyler der er sammenføjet, er sekvensidentitetsregionen mellem 150 og 300 basepar i længden.
17. Fremgangsmåde ifølge et hvilket som helst af kravene 1-16, som er en fremgangsmåde ti! indsættelse af et DNA-fragment af interesse i en linearise-ret vektor til at danne et cirkulært molekyle, yderligere omfattende tilføjelse af sekvenser ved PCR-amplifikation til hver ende af det intakte dobbeltstrengede DNA-molekyie, der er identiske med sekvenser på hver ende af den iinearise-rede vektor.
DK06813388.3T 2005-08-11 2006-08-11 In vitro rekombination fremgangsmåde DK1915446T3 (da)

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