EMULSION PCR AND AMPLICON CAPTURE
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.
1 . A method of attaching a polynucleotide to a surface comprising:
placing a hydrophilic compartment of an inverse emulsion comprising a polynucleotide and a surface, wherein said polynucleotide comprises a masked binding moiety and said surface comprises a binding moiety binding partner, under conditions suitable to activate said binding moiety which binds said binding partner, whereby said polynucleotide is attached to said surface.
2 . The method according to claim 1 , wherein said masked binding moiety is a caged biotin.
3 . The method according to claim 1 , wherein said masked binding moiety is a caged fluorescein.
4 . The method according to claim 1 , wherein said placing comprising exposing said compartment to electromagnetic radiation suitable to activate said binding moiety.
5 . The method according to claim 1 , wherein said masked binding moiety is attached to a nucleotide analog.
6 . The method according to claim 5 , wherein said nucleotide analog comprises isoC.
7 . The method according to claim 5 , wherein said nucleotide analog comprises isoG.
8 . The method according to claim 1 , wherein said polynucleotide is a primer.
9 . The method according to claim 1 , wherein said polynucleotide is an amplicon.
10 . The method according to claim 9 , wherein said polynucleotide comprises a label.
11 . The method according to claim 10 , wherein said label is digoxigenin.
12 . The method according to claim 1 , wherein said binding partner is an antibody.
13 . The method according to claim 1 , wherein said binding partner is streptavidin.
14 . The method according to claim 1 , wherein said binding partner is attached to a bead.
15 . A method of attaching an amplicon to a bead comprising:
(a) producing an amplicon in a hydrophilic compartment of an inverse emulsion comprising a template polynucleotide, a surface and reagents suitable for polynucleotide amplification, wherein said amplicon comprises a masked binding moiety and said surface comprises a binding moiety binding partner; and
(b) uncaging said masked binding moiety under conditions in which the uncaged moiety binds said binding partner whereby said amplicon is attached to said surface.
16 . The method according to claim 15 , wherein said masked binding moiety is a caged biotin.
17 . The method according to claim 15 , wherein said masked binding moiety is a caged fluorescein.
18 . The method according to claim 15 , wherein said uncaging comprising exposing said compartment to electromagnetic radiation suitable to uncage said masked binding moiety.
19 . The method according to claim 15 , wherein said amplicon is double stranded.
20 . The method according to claim 15 , wherein said amplicon is single-stranded.
21 . The method according to claim 15 , wherein said amplicon is single and/or double-stranded.
22 . The method according to claim 15 , wherein said amplicon comprises RNA.
23 . The method according to claim 15 , wherein said amplicon comprises DNA.
24 . The method according to claim 15 , wherein said amplicon is produced by PCR.
25 . The method according to claim 24 , wherein said PCR is asymmetric PCR.
26 . The method according to claim 24 , wherein said PCR is linear PCR.
27 . The method according to claim 15 , wherein said amplicon is produced by a single nucleotide extension.
28 . The method according to claim 15 , wherein said amplicon is produced by LCR.
29 . The method according to claim 15 , wherein said masked binding moiety is attached to a nucleotide analog.
30 . The method according to claim 29 , wherein said nucleotide analog is isoC.
31 . The method according to claim 29 , wherein said nucleotide analog is isoG.
32 . The method according to claim 15 , wherein said amplicon comprises a label.
33 . The method according to claim 32 , wherein said label is digoxigenin.
34 . The method according to claim 15 , wherein said binding partner is an antibody.
35 . The method according to claim 15 , wherein said binding partner is streptavidin.