DE602004036672C5 - Nukleinsäureamplifikation auf Basis von Kügelchenemulsion - Google Patents
Nukleinsäureamplifikation auf Basis von Kügelchenemulsion Download PDFInfo
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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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- 239000011324 bead Substances 0.000 title claims 18
- 230000003321 amplification Effects 0.000 title claims 3
- 238000003199 nucleic acid amplification method Methods 0.000 title claims 3
- 239000000839 emulsion Substances 0.000 title claims 2
- 150000007523 nucleic acids Chemical class 0.000 title 1
- 102000039446 nucleic acids Human genes 0.000 title 1
- 108020004707 nucleic acids Proteins 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract 9
- 108020004414 DNA Proteins 0.000 claims abstract 8
- 108091093088 Amplicon Proteins 0.000 claims 8
- 108010090804 Streptavidin Proteins 0.000 claims 3
- 239000000203 mixture Substances 0.000 claims 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims 1
- 238000012408 PCR amplification Methods 0.000 claims 1
- 238000009825 accumulation Methods 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 230000000295 complement effect Effects 0.000 claims 1
- 239000004530 micro-emulsion Substances 0.000 claims 1
- 238000004064 recycling Methods 0.000 claims 1
- 239000001226 triphosphate Substances 0.000 claims 1
- 235000011178 triphosphate Nutrition 0.000 claims 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims 1
- 238000012163 sequencing technique Methods 0.000 abstract 3
- 238000001712 DNA sequencing Methods 0.000 abstract 1
- 108091036333 Rapid DNA Proteins 0.000 abstract 1
- 238000009396 hybridization Methods 0.000 abstract 1
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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)...
Description
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Claims (5)
- 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.
- Verfahren nach Anspruch 1, bei dem in dem Schritt des Amplifizierens ein Polymerasekettenreaktion-Amplifizierungsprozess eingesetzt wird.
- Verfahren nach Anspruch 1, das ferner das Rezyklieren und Wiederverwenders der Kügelchen des zweiten Teilsatzes umfasst.
- 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.
- 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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