DK1907583T3 - Enkeltmolekyle-arrays til genetisk og kemisk analyse - Google Patents

Enkeltmolekyle-arrays til genetisk og kemisk analyse Download PDF

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DK1907583T3
DK1907583T3 DK06784802.8T DK06784802T DK1907583T3 DK 1907583 T3 DK1907583 T3 DK 1907583T3 DK 06784802 T DK06784802 T DK 06784802T DK 1907583 T3 DK1907583 T3 DK 1907583T3
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dna
probes
concatemers
target
array
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DK1907583T4 (da
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Radoje Drmanac
Matthew J Callow
Snezana Drmanac
Brian K Hauser
George Yeung
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Complete Genomics Inc
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Claims (18)

1. Tilfældigt array, der omfatter: et fast substrat med en overflade og et antal adskilte regioner anbragt i et regelmæssigt array på overfladen, interregionale områder mellem de adskilte regioner og enkeltstrengede DNA-concatemerer, der er tilfældigt indfanget på de adskilte regioner; hvor concatemererne er fikseret til de adskilte regioner ved en densitet, således at i det mindste størstedelen af concatemererne kan opløses optisk, hvor de adskilte regioner hver har et areal, der åbner mulighed for indfangning af ikke mere end en enkelt concatemer, og hvor de interregionale områder i det væsentlige er fri for concatemerer.
2. Tilfældigt array ifølge krav 1, hvor de adskilte regioner er anbragt i et retlinjet mønster.
3. Tilfældigt array ifølge et hvilket som helst af krav 1 eller 2, hvor antallet af concatemerer er immobiliseret på de adskilte regioner ved hjælp af tiltrækkende ikke-kovalente interaktioner.
4. Tilfældigt array ifølge krav 3, hvor de tiltrækkende ikke-kovalente interaktioner er udvalgt blandt van der Waals-kræfter, hydrogenbinding, ioniske interaktioner og hydrofobe interaktioner.
5. Tilfældigt array ifølge et hvilket som helst af kravene 1 til 4, hvor overfladen omfatter kiselsyre.
6. Tilfældigt array ifølge krav 1, hvor de adskilte regioner omfatter funktionelle dele.
7. Tilfældigt array ifølge krav 1, hvor overfladen er aminmodificeret.
8. Tilfældigt array ifølge krav 1, hvor arealet af de adskilte regioner er i intervallet fra 0,04 ym2 til 1 ym2.
9. Tilfældigt array ifølge krav 1, hvor concatemererne har en størrelse i intervallet 50-250 kb.
10. Tilfældigt array ifølge krav 1, hvor en projektion af den tilfældige spiraltilstand af concatemererne på en overflade i det væsentlige svarer til arealet af de adskilte regioner til muliggørelse af indfangning af ikke mere end en enkelt concatemer i hver region.
11. Fremgangsmåde til bestemmelse af en sekvens af et målpolynukleotid, hvilken fremgangsmåde omfatter: (a) frembringelse af et antal målconcatemerer fra målpolynukleotidet, hvor hver målconcatemer omfatter flere kopier af et fragment af målpolynukleotidet, og antallet af målconcatemerer indbefatter et antal fragmenter, der i det væsentlige dækker målpolynukleotidet; (b) fremstilling af et tilfældigt array af målconcatemerer, der er fikseret til en overflade ved en densitet, således at i det mindste størstedelen af målconcatemererne kan opløses optisk; (c) identificering af en sekvens af mindst en del af hvert fragment i hver målconcatemer; og (d) rekonstruering af en nukleotidsekvens for målpolynukleotidet, hvor det tilfældige array er et array ifølge krav 1 eller krav 2 .
12. Fremgangsmåde ifølge krav 11, hvor identificeringen omfatter: (i) hybridisering af en eller flere probr fra et første sæt af prober til det tilfældige array under betingelser, der åbner mulighed for dannelse af perfekt parrede duplekser mellem den ene eller de flere prober og komplementære sekvenser på målconcatemererne; (ii) hybridisering af en eller flere prober fra et andet sæt af prober til det tilfældige array under betingelser, der åbner mulighed for dannelse af perfekt parrede duplekser mellem den ene eller de flere prober og komplementære sekvenser på målconcatemererne; (iii) ligering af prober fra det første og andet sæt, der er hybridiseret til en målconcatemer på fortløbende steder; (iv) identificering af sekvenserne af de ligerede første og anden prober; og (v) gentagelse af trin (i) til (iv) til derved bestemmelse af en sekvens for polynukleotidet.
13. Fremgangsmåde ifølge krav 12, hvor en eller flere prober fra det første sæt eller fra det andet sæt omfatter et påviseligt mærke.
14. Fremgangsmåde ifølge krav 13, hvor det påviselige mærke omfatter et fluorescerende farvestof.
15. Fremgangsmåde ifølge et hvilket som helst af kravene 12 til 14, hvor den ene eller de flere prober fra det første sæt omfatter et eller flere degenererede nukleotider.
16. Fremgangsmåde ifølge et hvilket som helst af kravene 12 til 15, hvor en eller flere prober fra det andet sæt af prober omfatter et eller flere degenererede nukleotider.
17. Fremgangsmåde til fremstilling af et tilfældigt array af enkeltstrengede DNA-concatemerer, som omfatter: (a) tilvejebringelse af et fast substrat, der omfatter en overflade, hvor overfladen omfatter et regelmæssigt array af adskilte regioner, hvortil DNA-concatemerer kan binde, der er adskilt af interregionale områder, hvortil DNA-concatemerer ikke binder; hvor de adskilte regioner hver har et areal, der åbner mulighed for indfangning af ikke mere end en enkelt concatemer; (b) deponering på overfladen af en sammensætning, der omfatter et antal enkeltstrengede DNA-concatemerer, og muliggørelse af, at concatemererne kan binde i de adskilte regioner.
18. Fremgangsmåde ifølge krav 17, hvor en projektion af den tilfældige spiraltilstand af concatemererne på en overflade i det væsentlige svarer til arealet af de adskilte regioner.
DK06784802.8T 2005-06-15 2006-06-13 Enkeltmolekyle-arrays til genetisk og kemisk analyse DK1907583T4 (da)

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US69077105P 2005-06-15 2005-06-15
US72511605P 2005-10-07 2005-10-07
US77641506P 2006-02-24 2006-02-24
PCT/US2006/022895 WO2006138257A2 (en) 2005-06-15 2006-06-13 Single molecule arrays for genetic and chemical analysis

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DK12180882.8T DK2620510T4 (da) 2005-06-15 2006-06-13 Enkeltmolekyle-arrays til genetisk og kemisk analyse
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