Compositions and methods for analyzing isolated polynucleotides

• US 7,604,940 B1
• Filed: 03/15/2006
• Issued: 10/20/2009
• Est. Priority Date: 03/16/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Voss, Karl
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/375,824
• Time in Patent Office: 1,315 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Compositions and methods of use are disclosed for the analysis of polynucleotides isolated in individual reaction compartments.

Claims

1. A method of analyzing the contents of a microcapsule comprising inserting the microcapsule into a capillary of a capillary electrophoresis system, and analyzing one or more molecules released from the microcapsule in the capillary.

2. The method according to claim 1, wherein said molecules are produced by sequencing one or more polynucleotides contained within said microcapsule.

3. The method according to claim 1, wherein said one or more molecules are produced by sequencing one or more polynucleotides contained within an aqueous compartment of an inverse emulsion and said microcapsule is produced by encapsulating said compartment.

4. The method according to claim 2, wherein said sequencing is Sanger sequencing.

5. The method according to claim 2, wherein said one or more polynucleotides are attached to a surface.

6. The method according to claim 2, wherein said polynucleotides comprise amplicons produced by clonally amplifying a target polynucleotide.

7. The method according to claim 6, wherein said target polynucleotide is clonally amplified in a second microcapsule.

8. The method according to claim 6, wherein said target polynucleotide is clonally amplified in an aqueous compartment of an inverse emulsion.

9. The method according to claim 1, wherein said microcapsule comprises a hydrogel.

10. The method according to claim 1, wherein said microcapsule is a colloidosome.

11. The method according to claim 1, wherein said microcapsule is produced by layer-by-layer polymer deposition.

12. The method according to claim 1, wherein said microcapsule is produced by polymer precipitation.

13. A method of analyzing the contents of a plurality of microcapsules comprising inserting the plurality of microcapsules into a plurality of capillaries of an electrophoresis system, and analyzing the contents of the capsules.

14. A sequencing method comprising:

a) clonally amplifying a plurality of target polynucleotides in a plurality of aqueous compartments of an inverse emulsion, wherein the clonally amplified target polynucleotides are derived from a single polynucleotide molecule; and

b) sequencing the amplification products of a) in a plurality of microcapsules.

15. The method according to claim 14, further comprising:

c) analyzing the sequencing products of b) in a capillary electrophoresis system.

16. The method according to claim 15, wherein said microcapsules are inserted into the capillaries of said system and said sequencing products are released from said microcapsules into said capillaries.

17. The method according to claim 14, wherein at least one of said microcapsules comprises a hydrogel.

18. The method according to claim 14, wherein at least one of said microcapsules is a colloidosome.

19. The method according to claim 14, wherein at least one of said microcapsules is produced by layer-by-layer polymer deposition.

20. The method according to claim 14, wherein at least one of said microcapsules is produced by polymer deposition.

21. The method according to claim 14, wherein said sequencing is Sanger sequencing.

22. The method according to claim 14, wherein said sequencing is pyrosequencing.