DE602004036672C5 - Nukleinsäureamplifikation auf Basis von Kügelchenemulsion - Google Patents

Nukleinsäureamplifikation auf Basis von Kügelchenemulsion Download PDF

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DE602004036672C5
DE602004036672C5 DE602004036672T DE602004036672T DE602004036672C5 DE 602004036672 C5 DE602004036672 C5 DE 602004036672C5 DE 602004036672 T DE602004036672 T DE 602004036672T DE 602004036672 T DE602004036672 T DE 602004036672T DE 602004036672 C5 DE602004036672 C5 DE 602004036672C5
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bead
amplicon
immobilized
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Maithreyan Srinivasan
Yi-Ju Chen
Michael Weiner
Steven Lefkowitz
Kenton Lohman
Gary J. Sarkis
John H. Leamon
Jan Berka
Jonathan Rothberg
Vinod Makhijani
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454 Life Science Corp
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Abstract

Verfahren zum Anreichern eines Amplikons, umfassend: (a) Verteilen einer Amplifikationslösung, die ein ausreichendes Gemisch von Reagentien, die zur Durchführung einer Amplifikationsreaktion der Templat-DNA erforderlich sind, eine Mehrzahl von Kügelchen und eine Mehrzahl von Arten von Templat-DNAs umfasst, in einer Mehrzahl von wässrigen Mikroemulsionströpfchen in einer wärmestabilen Ölphase, wobei ein erster Teilsatz der Tröpfchen ein Kügelchen und eine oder mehrere der Arten von Templat-DNAs darin eingekapselt umfasst, und ein zweiter Teilsatz der Tröpfchen ein Kügelchen ohne irgendeine der Arten von Templat-DNA darin eingekapselt umfasst, (b) Amplifizieren der Arten von Templat-DNAs innerhalb des ersten Teilsatzes von Tröpfchen, wobei das Kügelchen innerhalb des ersten Teilsatzes von wässrigen Tröpfchen ein daran immobilisiertes Amplikon aufweist, (c) Aufbrechen der wässrigen Emulsionströpfchen zum Freisetzen der Kügelchen des ersten und des zweiten Teilsatzes, (d) Hybridisieren eines biotinylierten Primers an das Kügelchen-immobilisierte Amplikon, der zu dem 3'-Ende des Amplikons komplementär ist und in Schritt (e)...

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Claims (5)

  1. Verfahren zum Anreichern eines Amplikons, umfassend: (a) Verteilen einer Amplifikationslösung, die ein ausreichendes Gemisch von Reagentien, die zur Durchführung einer Amplifikationsreaktion der Templat-DNA erforderlich sind, eine Mehrzahl von Kügelchen und eine Mehrzahl von Arten von Templat-DNAs umfasst, in einer Mehrzahl von wässrigen Mikroemulsionströpfchen in einer wärmestabilen Ölphase, wobei ein erster Teilsatz der Tröpfchen ein Kügelchen und eine oder mehrere der Arten von Templat-DNAs darin eingekapselt umfasst, und ein zweiter Teilsatz der Tröpfchen ein Kügelchen ohne irgendeine der Arten von Templat-DNA darin eingekapselt umfasst, (b) Amplifizieren der Arten von Templat-DNAs innerhalb des ersten Teilsatzes von Tröpfchen, wobei das Kügelchen innerhalb des ersten Teilsatzes von wässrigen Tröpfchen ein daran immobilisiertes Amplikon aufweist, (c) Aufbrechen der wässrigen Emulsionströpfchen zum Freisetzen der Kügelchen des ersten und des zweiten Teilsatzes, (d) Hybridisieren eines biotinylierten Primers an das Kügelchen-immobilisierte Amplikon, der zu dem 3'-Ende des Amplikons komplementär ist und in Schritt (e) durch ein Streptavidin-beschichtetes Kügelchen zur Bildung eines Biotin-Streptavidin-Einfang-Ziel-Paars immobilisiert wird, (e) Bilden eines Komplex aus Amplikon-immobilisierten Kügelchen und Anreicherungskügelchen, wobei das Streptavidin-beschichtete Anreicherungskügelchen, welches für die Anziehungskraft eines Magnetfeldes empfänglich ist, und der hybridisierte biotinylierte Primer ein Biotin-Streptavidin-Einfang-Ziel-Paar bilden, und (f) Anwenden eines Magnetfeldes zum Isolieren des Kügelchen-Komplexes mit einer Anreicherung von Amplikon-immobilisierten Kügelchen von solchen Kügelchen, die kein gebundenes Amplikon aufweisen.
  2. Verfahren nach Anspruch 1, bei dem in dem Schritt des Amplifizierens ein Polymerasekettenreaktion-Amplifizierungsprozess eingesetzt wird.
  3. Verfahren nach Anspruch 1, das ferner das Rezyklieren und Wiederverwenders der Kügelchen des zweiten Teilsatzes umfasst.
  4. Verfahren nach Anspruch 1, das ferner das Verlängern des an das 3'-Ende des Amplikons hybridisierten biotinylierten Primers von Schritt (d) durch Zusetzen von DNA-Polymerase und der vier natürlichen Desoxynukleotid-Triphosphate (dNTPs) zu dem Kügelchengemisch umfasst, wobei die Verlängerung die Bindung zwischen dem biotinylierten Primer und dem Kügelchen-immobilisierten Amplikon verstärkt.
  5. Verfahren nach Anspruch 1, wobei das Verfahren ferner umfasst: Trennen des biotinylierten Primers von dem Kügelchen-immobilisierten Amplikon zum Trennen der Streptavidin-beschichteten Anreicherungskügelchen von dem Amplikon-immobilisierten Kügelchen.
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