Emulsion PCR and amplicon capture
First Claim
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1. A method of attaching a polynucleotide to a surface comprising:
- generating a hydrophilic compartment of an inverse emulsion comprising a polynucleotide and a surface,wherein said-polynucleotide comprises an isoC or isoG to which a photoactivatable binding moiety and said surface comprises a binding moiety binding partner, andactivating said binding moiety by exposing said compartment to activating light, which activated binding moiety binds to said binding partner, whereby said polynucleotide is attached to said surface.
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Abstract
Compositions and methods of use are disclosed for clonally amplifying target polynucleotide sequences in solution and attaching the amplicons to a surface by activation of a masked binding moiety. In an embodiment, the amplicons comprise the masked binding moiety and the surface comprises a binding partner of the binding moiety. Upon activation of the binding moiety, the amplicons bind to the binding partner on the surface. In a non-limiting example, the masked binding moiety is caged biotin or caged fluorescein, while the corresponding binding partner is avidin or an anti-fluorescein antibody.
81 Citations
32 Claims
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1. A method of attaching a polynucleotide to a surface comprising:
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generating a hydrophilic compartment of an inverse emulsion comprising a polynucleotide and a surface, wherein said-polynucleotide comprises an isoC or isoG to which a photoactivatable binding moiety and said surface comprises a binding moiety binding partner, and activating said binding moiety by exposing said compartment to activating light, which activated binding moiety binds to said binding partner, whereby said polynucleotide is attached to said surface. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of attaching an amplicon to a bead comprising:
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(a) producing an amplicon in a hydrophilic compartment of an inverse emulsion comprising a template polynucleotide, a bead having a surface and reagents suitable for polynucleotide amplification, wherein said amplicon comprises an isoC or isoG to which a photoactivatable binding moiety and said surface comprises a binding moiety binding partner; and (b) activating said binding moiety by exposing said compartment to activating light, which activated binding moiety binds said binding partner whereby said amplicon is attached to said surface of said bead. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification