Emulsion PCR and amplicon capture

• US 8,293,502 B2
• Filed: 04/14/2011
• Issued: 10/23/2012
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Schroeder, Benjamin
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US13/087,278
• Publication Number:
  US 20110257046A1
• Time in Patent Office: 558 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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. 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.

Claims

1. A method of attaching a polynucleotide to a surface comprising:

(a) generating a hydrophilic compartment of an inverse emulsion comprising a polynucleotide and a surface, wherein said polynucleotide comprises a photoactivatable binding moiety and said surface comprises a binding moiety binding partner; and

(b) exposing said hydrophilic compartment to activating electromagnetic radiation to activate said photoactivatable binding moiety to bind said binding partner thereby attaching said polynucleotide to said surface.

2. The method according to claim 1, wherein said photoactivatable binding moiety is a caged biotin.

3. The method according to claim 1, wherein said photoactivatable binding moiety is a caged fluorescein.

4. The method according to claim 1, wherein said polynucleotide comprises isoC to which said binding moiety is attached.

5. The method according to claim 1, wherein said polynucleotide comprises isoG to which said binding moiety is attached.

6. The method according to claim 1, wherein said polynucleotide is a primer.

7. The method according to claim 1, wherein said polynucleotide is an amplicon.

8. The method according to claim 7, wherein said polynucleotide comprises a label.

9. The method according to claim 8, wherein said label is digoxigenin.

10. The method according to claim 1, wherein said binding partner is an antibody.

11. The method according to claim 1, wherein said binding partner is streptavidin.

12. The method according to claim 1, wherein said binding partner is attached to a bead.

13. A method of attaching an amplicon to a bead comprising:

(a) amplifying a template polynucleotide in a hydrophilic compartment of an inverse emulsion to produce an amplicon, wherein the hydrophilic compartment comprises a template polynucleotide, a surface and reagents suitable for polynucleotide amplification, wherein said amplicon comprises a photoactivatable binding moiety and said surface comprises a binding moiety binding partner; and

(b) activating said photoactivatable binding moiety by exposing said compartment to activating electromagnetic radiation to activate said binding moiety to bind said binding partner thereby attaching said amplicon to said surface.

14. The method according to claim 13, wherein said photoactivatable binding moiety is a caged biotin.

15. The method according to claim 13, wherein said photoactivatable binding moiety is a caged fluorescein.

16. The method according to claim 13, wherein said amplicon is double stranded.

17. The method according to claim 13, wherein said amplicon is single-stranded.