EMULSION PCR AND AMPLICON CAPTURE

• US 20080076909A1
• Filed: 06/14/2007
• Published: 03/27/2008
• Est. Priority Date: 06/30/2006
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: SCHROEDER, Benjamin
• Application Number:
  US11/763,410
• Publication Number:
  US 20080076909A1
• Time in Patent Office: Days
• US Class Current: 536/25.3
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

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

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.