US2008076909A1PendingUtilityA1

Emulsion pcr and amplicon capture

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Assignee: APPLERA CORPPriority: Jun 30, 2006Filed: Jun 14, 2007Published: Mar 27, 2008
Est. expiryJun 30, 2026(expired)· nominal 20-yr term from priority
Inventors:Benjamin G. Schroeder
C12Q 1/6834B01J 2219/00605C40B 30/04B01J 2219/0063B01J 2219/00612B01J 2219/00637B01J 2219/0061C40B 80/00B01J 2219/00648C40B 40/06
67
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Claims

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.

Claims

exact text as granted — not AI-modified
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.

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