DE10184524T1 - DNA Sequenzierung durch parallele Erweiterung von Oligonukleotiden - Google Patents

DNA Sequenzierung durch parallele Erweiterung von Oligonukleotiden Download PDF

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DE10184524T1
DE10184524T1 DE10184524T DE10184524T DE10184524T1 DE 10184524 T1 DE10184524 T1 DE 10184524T1 DE 10184524 T DE10184524 T DE 10184524T DE 10184524 T DE10184524 T DE 10184524T DE 10184524 T1 DE10184524 T1 DE 10184524T1
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polynucleotide
sequence
probe
ligated
oligonucleotide
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Stephen Calif. Macevicz
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Solexa Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/582—Recycling of unreacted starting or intermediate materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

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Abstract

Verfahren zum Identifizieren einer Sequenz von Nukleotiden in einem Polynukleotid, wobei das Verfahren umfasst: (a) Bereitstellen eines ersten Matrizen-Polynukleotids umfassend eine Binderegion, die eine bekannte Sequenz beinhaltet und eine Zielregion, die eine unbekannte Sequenz beinhaltet; (b) Verlängern verschiedener Sequenz-initialisierender Oligonukleotide durch wiederholte Zyklen einer Duplex-Verlängerung entlang einer Matrize, wobei das Verfahren zur Verlängerung eines bestimmten initialisierenden Oligonukleotids umfasst: Bilden eines Duplex zwischen dem ersten Polynukleotid, das in Schritt (a) bereitgestellt wurde und einem zweiten Polynukleotid, das eine Sequenz des initialisierenden Oligonukleotids enthält, wobei das initialisierende Oligonukleotid mit einem Teil der Binderegion des ersten Polynukleotids hybridisiert und Durchführen von wiederholten Zyklen der Duplex-Verlängerung, die beinhalten: 1. Bereitstellen einer Vielzahl an verschiedenen Oligonukleotidsonden mit unterschiedlichen Sequenzen; 2. Verlängern des zweiten Polynukleotids durch Ligieren von einem der verschiedenen Oligonukleotidsonden mit dem zweiten Polynukleotid, um ein längeres zweites Polynukleotid zu erzeugen, wobei die Sonde der Vielzahl an verschiedenen...

Claims (21)

  1. Verfahren zum Identifizieren einer Sequenz von Nukleotiden in einem Polynukleotid, wobei das Verfahren umfasst: (a) Bereitstellen eines ersten Matrizen-Polynukleotids umfassend eine Binderegion, die eine bekannte Sequenz beinhaltet und eine Zielregion, die eine unbekannte Sequenz beinhaltet; (b) Verlängern verschiedener Sequenz-initialisierender Oligonukleotide durch wiederholte Zyklen einer Duplex-Verlängerung entlang einer Matrize, wobei das Verfahren zur Verlängerung eines bestimmten initialisierenden Oligonukleotids umfasst: Bilden eines Duplex zwischen dem ersten Polynukleotid, das in Schritt (a) bereitgestellt wurde und einem zweiten Polynukleotid, das eine Sequenz des initialisierenden Oligonukleotids enthält, wobei das initialisierende Oligonukleotid mit einem Teil der Binderegion des ersten Polynukleotids hybridisiert und Durchführen von wiederholten Zyklen der Duplex-Verlängerung, die beinhalten: 1. Bereitstellen einer Vielzahl an verschiedenen Oligonukleotidsonden mit unterschiedlichen Sequenzen; 2. Verlängern des zweiten Polynukleotids durch Ligieren von einem der verschiedenen Oligonukleotidsonden mit dem zweiten Polynukleotid, um ein längeres zweites Polynukleotid zu erzeugen, wobei die Sonde der Vielzahl an verschiedenen Sonden, die ligiert wird, von der Sequenz des ersten Polynukleotids abhängt und wobei die ligierte Sonde nicht fähig ist mit einer anderen der Oligonukleotidsonden, die in Schritt (b)(1) bereitgestellt wurden, zu ligieren; 3. Detektieren einer Markierung, die mit der Sonde, die in Schritt (b)(2) ligiert wurde, assoziiert ist, wobei die Markierung Basen der ligierten Probe entspricht, aber nicht der gesamten Sequenz der ligierten Probe; und 4. Neubilden eines verlängerbaren Endes auf dem zweiten Polynukleotid, so dass zusätzliche Sonden an das zweite Polynukleotid in nachfolgenden Verlängerungsrunden des zweiten Polynukleotids angebracht werden können; und (c) Bestimmen einer fortlaufenden Basensequenz der unbekannten Sequenz basierend auf der Kombination der Reihen an Verlängerungen der verschiedenen initialisierenden Oligonukleotide.
  2. Verfahren nach Anspruch 1, wobei Schritt (b)(4) Spalten der Sonde, die in Schritt (b)(2) ligiert wurde, zwischen zwei Nukleotiden, die innen in der Sonde liegen, umfasst.
  3. Verfahren nach Anspruch 2, wobei das Spalten der Sonde zwischen zwei Nukleotiden, die innen in der Sonde liegen, auch die Markierung von dem zweiten Polynukleotid entfernt.
  4. Verfahren nach einem der Ansprüche 1–3, wobei die Reihen an Verlängerungen der einzelnen initialisierenden Oligonukleotide aus Schritt (b) weiter das Versehen eines zweiten Polynukleotids mit einer Kappe umfasst, immer wenn das zweite Polynukleotid nicht mit einer Oligonukleotidsonde in Schritt (b)(2) ligiert.
  5. Verfahren nach einem der Ansprüche 1–4, wobei mindestens einige der Sonden, die in Schritt (b)(1) bereitgestellt wurden, mindestens eine Desoxyinosin-Base umfassen.
  6. Verfahren nach einem der Ansprüche 1–5, wobei die Sonden, die in Schritt (b)(1) bereitgestellt wurden, eine vorbestimmte Länge ausgewählt aus 8 Nukleotiden, 9 Nukleotiden und 12 Nukleotiden aufweisen.
  7. Verfahren nach einem der Ansprüche 1–6, wobei die initialisierenden Oligonukieotide versetzt („offset”) voneinander sind.
  8. Verfahren nach Anspruch 7, wobei die initialisierenden Oligonukleotide, die zum Bestimmen der Sequenz der fortlaufenden Basen der unbekannten Sequenz verwendet werden, durch eine Base voneinander versetzt sind.
  9. Verfahren zum Identifizieren einer Sequenz von Nukleotiden in einem ersten Polynukleotid, wobei das Verfahren die Schritte umfasst: (a) Verlängern eines initialisierenden Oligonukleotids entlang des ersten Polynukleotids durch Ligieren einer Oligonukleotidsonde daran, um ein verlängertes Duplex zu bilden, das nicht fähig ist mit einer weiteren Oligonukleotidsonde zu ligieren; (b) Detektieren einer Markierung, die mit der Oligonukleotidsonde, die daran ligiert ist, assoziiert ist; (c) Neubilden eines verlängerbaren Endes auf dem verlängerten Duplex durch Spalten des verlängerten Duplex an einer Position zwischen zwei Basen der Oligonukleotidsonde, die daran ligiert ist; und (d) Wiederholen der Schritte (a)–(c) bis die Sequenz der Nukleotide bestimmt ist.
  10. Verfahren nach einem der Ansprüche 1–9, wobei die Markierung an ein End-Nukleotid der ligierten Sonde gegenüber von dem Ende der ligierten Sonde, das ligiert wird, angebracht ist.
  11. Verfahren nach einem der Ansprüche 1–10 weiter umfassend Amplifizieren des ersten Polynukleotids, um das erste Polynukleotid, das in Schritt (b) verwendet wird, zu erzeugen.
  12. Verfahren nach Anspruch 11, wobei das amplifizierte erste Polynukleotid mehrere Kopien des bereitgestellten ersten Nukleotids, das auf einem Kügelchen getragen wird, umfasst.
  13. Verfahren nach einem der Ansprüche 1–12 weiter umfassend Bestimmen einer fortlaufenden Basensequenz von verschiedenen unbekannten Sequenzen verschiedener Polynukleotide basierend auf der Durchführung mehrerer Verlängerungszyklen, Identifizierungs- und Neubildungsschritte an mehreren verschiedenen ersten Polynukleotiden nebeneinander.
  14. Verfahren nach Anspruch 13, wobei die mehreren verschiedenen ersten Polynukleotide eine bekannte Sequenz in der Binderegion miteinander gemeinsam haben.
  15. Verfahren nach einem der Ansprüche 1–14 weiter umfassend Bestimmen der Reihenfolge jedes Nukleosids in dem Polynukleotid, das sequenziert wird.
  16. Verfahren nach einern der Ansprüche 1–15, wobei die initialisierenden Oligonukleotide mindestens 20 Basen lang sind und die Binderegion mindestens 20 Basen lang ist und lang genug ist, um einen Startpunkt für eine Reihe an verschiedenen Sequenz-initialisierenden Oligonukleotiden bereitzustellen.
  17. Verfahren nach Anspruch 9, wobei Schritt (b) weiter Bereitstellen mehrerer verschiedener Oligonukleotidsonden umfasst, wobei eine der bereitgestellten Sonden an das initialisierende Oligonukleotid ligiert ist, um ein verlängertes Duplex zu bilden.
  18. Verfahren nach Anspruch 17, wobei mindestens einige der bereitgestellten Sonden mindestens eine Desoxyinosin-Base umfassen.
  19. Verfahren nach Anspruch 17 oder Anspruch 18, wobei die bereitgestellten Sonden eine vorbestimmte Länge ausgewählt aus 8 Nukleotiden, 9 Nukleotiden und 12 Nukleotiden aufweisen.
  20. System zur Durchführung des Verfahrens nach einem der Ansprüche 1–19, wobei das System umfasst: eine Reihe an Reagenzbehältern, die Reagenzien zur Durchführung des Verfahrens enthalten; mindestens ein Reaktionsgefäß, das die ersten Polynukleotide, die an einen Festphasenträger gebunden sind, enthält; eine computergesteuerte Vorrichtung, die ausgelegt ist, um, in wiederholten Zyklen, Ausgeben von markierten Sonden und Ligationsreagenzien in das Reaktionsgefäß zu steuern, um eine der Sonden an ein Polynukleotid, das das initialisierende Oligonukleotid enthält, zu ligieren; und um Ausgeben der Spaltungsreagenzien in das Reaktionsgefäß zu steuern, wobei die Spaltungsreagenzien ausgelegt sind, um die Verknüpfung zwischen Basen der ausgegebenen markierten Sonden zu spalten, um ein verlängerbares Ende neu zu bilden; und eine Detektionsstation, die ausgelegt ist, um, in wiederholten Zyklen, die Markierung, die mit der ligierten Sonde assoziiert ist, zu detektieren.
  21. System nach Anspruch 20 weiter umfassend einen Prozessor, der ausgelegt ist, um eine fortlaufende Basensequenz aus den wiederholten Zyklen der Detektion der Markierungen zu bestimmen.
DE10184524T 1995-04-17 1996-04-16 DNA Sequenzierung durch parallele Erweiterung von Oligonukleotiden Pending DE10184524T1 (de)

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US08/424,663 US5750341A (en) 1995-04-17 1995-04-17 DNA sequencing by parallel oligonucleotide extensions
US424663 1995-04-17
EP10184524A EP2298786A1 (de) 1995-04-17 1996-04-16 DNA Sequenzierung durch parallele Erweiterung von Oligonukleotiden

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EP (5) EP2433950A1 (de)
JP (5) JP4546582B2 (de)
AU (1) AU718937B2 (de)
CA (1) CA2218017C (de)
DE (1) DE10184524T1 (de)
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US5750341A (en) 1998-05-12
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