DK2787565T3 - Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer - Google Patents

Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer Download PDF

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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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dna
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sequence
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Jerome Jendrisak
Gary Dahl
Haiying Li Grunenwald
Nicholas Caruccio
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Epict Technologies Corp
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    • C12N15/1034—Isolating an individual clone by screening libraries
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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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.
DK14171077.2T 2008-10-24 2009-10-24 Transposon-ende-sammensætninger og fremgangsmåder til at modificere nukleinsyrer DK2787565T3 (da)

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US10832608P 2008-10-24 2008-10-24
US10832108P 2008-10-24 2008-10-24
US10832908P 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

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DK09822831.5T DK2376517T3 (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
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