DK2787565T3 - Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer - Google Patents
Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer Download PDFInfo
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- DK2787565T3 DK2787565T3 DK14171077.2T DK14171077T DK2787565T3 DK 2787565 T3 DK2787565 T3 DK 2787565T3 DK 14171077 T DK14171077 T DK 14171077T DK 2787565 T3 DK2787565 T3 DK 2787565T3
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- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N9/1241—Nucleotidyltransferases (2.7.7)
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
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Claims (14)
1. Fremgangsmåde til parallel sekvensering af et mål-DNA, hvilken fremgangsmåde omfatter: a) tilvejebringelse af et bibliotek af lineære mål-DNA-ffagmenter omfattende en første transposon-ende og et første amplifikations-tag-domæne, der er fæstnet til 5’-ende af mål-DNA-ffagmenteme, og en anden transposon-ende og et andet amplifikations- tag-domæne, der er fæstnet til 5’-ende af den komplementære streng af mål-DNA-ffagmenteme, hvor transposon-endeme er fæstnet til DNA-ffagmenteme via en transpositionsreaktion in vitro omfattende en flerhed af transposomer, hvor hvert transposom omfatter en transposase og transposon-ender, b) denaturering af biblioteket af lineære DNA-ffagmenter for at generere enkeltstrengede DNA-ffagmenter, c) indfangning af de enkeltstrengede DNA-ffagmenter på et substrat på betingelser, hvor en del af ffagmenteme indfanges på substratet, og d) parallel sekvensering af de indfangede DNA-ffagmenter, hvor de indfangede DNA-ffagmenter sekvenseres samtidigt i et enkelt multiplexformat.
2. Fremgangsmåde ifølge krav 1, hvor de lineære DNA-fragmenter endvidere omfatter ét eller flere af et fangst-tag-domæne, et adresse-tag-domæne, et sekvenserings-tag-domæne og et restriktionssted-tag-domæne.
3. Fremgangsmåde ifølge et hvilket som helst af kravene 1 og 2, hvor de lineære DNA-fragmenter endvidere omfatter et adresse-tag-domæne eller et sekvenserings-tag-domæne.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 3, hvor denaturering er varmedenaturering.
5. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 4, hvor én eller begge transposon-ender omfatter et fangst-tag-domæne og substratet omfatter fangstmolekyler, der binder fangst-tag-domænet.
6. Fremgangsmåde ifølge krav 5, hvor fangstmolekylet er en DNA’sekvens, der er fæstnet til substratet.
7. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 6, hvor transposasen er en Tn5-transposase og transposon-endeme er Tn5-transposon-ender.
8. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 6, hvor transposasen er en Mu-transposase og transposon-endeme er Mu-transposon-ender.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 8, hvor substratet omfatter mindst en million forskellige indfangede DNA-fragmenter.
10. Fremgangsmåde ifølge et hvilket som helst af kravene 1 til 9, hvor mål-DNA’et omfatter en flerhed af forskellige nukleinsyrer.
11. Fremgangsmåde til sekvensering af en målnukleinsyre, hvilken fremgangsmåde omfatter: (a) etablering af kontakt mellem målnukleinsyren og en flerhed af transposomer omfattende en transposase og en transposon-endesekvens, hvorved der opnås en flerhed af fragmenterede nukleinsyrer omfattende en transposon-endesekvens; (b) udvidelse af en primer hybridiseret til hver transposon-endesekvens af flerheden af fragmenterede nukleinsyrer, hvor primeren omfatter et fangst-tag; (c) indfangning af de udvidede nukleinsyrer via fangst-tag’et med en flerhed af fangstsonder bundet til en overflade og (d) samtidig sekvensering af de indfangede nukleinsyrer i et enkelt multiplexformat.
12. Fremgangsmåde ifølge krav 11, hvor trin (b) omfatter amplifikation af flerheden af fragmenterede nukleinsyrer.
13. Fremgangsmåde ifølge krav 11, hvor overfladen omfatter mindst en million forskellige indfangede nukleinsyrer.
14. Fremgangsmåde ifølge krav 11, hvor målnukleinsyren omfatter en flerhed af forskellige nukleinsyrer.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10832108P | 2008-10-24 | 2008-10-24 | |
US10832908P | 2008-10-24 | 2008-10-24 | |
US10832608P | 2008-10-24 | 2008-10-24 | |
US15286809P | 2009-02-16 | 2009-02-16 | |
US15543109P | 2009-02-25 | 2009-02-25 | |
US18453009P | 2009-06-05 | 2009-06-05 | |
EP13180374.4A EP2664678B1 (en) | 2008-10-24 | 2009-10-24 | Transposon end compositions and methods for modifying nucleic acids |
Publications (1)
Publication Number | Publication Date |
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DK2787565T3 true DK2787565T3 (da) | 2015-10-12 |
Family
ID=42119714
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DK09822831.5T DK2376517T3 (da) | 2008-10-24 | 2009-10-24 | Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer |
DK14171077.2T DK2787565T3 (da) | 2008-10-24 | 2009-10-24 | Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer |
DK17171687.1T DK3272879T3 (da) | 2008-10-24 | 2009-10-24 | Transposonendesammensætninger og fremgangsmåder til modificering af nukleinsyrer |
DK15179339.5T DK2963709T3 (da) | 2008-10-24 | 2009-10-24 | Transposon-ende-sammensætninger og fremgangsmåder til modificering af nukleinsyrer |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DK09822831.5T DK2376517T3 (da) | 2008-10-24 | 2009-10-24 | Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
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DK17171687.1T DK3272879T3 (da) | 2008-10-24 | 2009-10-24 | Transposonendesammensætninger og fremgangsmåder til modificering af nukleinsyrer |
DK15179339.5T DK2963709T3 (da) | 2008-10-24 | 2009-10-24 | Transposon-ende-sammensætninger og fremgangsmåder til modificering af nukleinsyrer |
Country Status (13)
Country | Link |
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EP (6) | EP2787565B1 (da) |
JP (1) | JP5794572B2 (da) |
CN (2) | CN102264914B (da) |
CA (1) | CA2750054C (da) |
DK (4) | DK2376517T3 (da) |
ES (5) | ES2743029T3 (da) |
HK (6) | HK1163107A1 (da) |
HU (1) | HUE027961T2 (da) |
LT (1) | LT2963709T (da) |
PL (1) | PL2963709T3 (da) |
PT (2) | PT2787565E (da) |
SI (1) | SI2963709T1 (da) |
WO (1) | WO2010048605A1 (da) |
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