Separation of pyrophosphate release and pyrophosphate detection

• US 8,765,419 B2
• Filed: 04/29/2013
• Issued: 07/01/2014
• Est. Priority Date: 02/25/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Hirschbein, Bernard, Gorpe-Yasar, Filiz
• Primary Examiner(s): Gross, Christopher M
• Assistant Examiner(s): Manteuffel, Richard L
• Application Number:
  US13/872,812
• Publication Number:
  US 20130237434A1
• Time in Patent Office: 428 Days
• Field of Search: 506/7, 506/16
• US Class Current: 435/91.5
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

The present technology relates to methods and systems for detection of pyrophosphate. As such, disclosed herein are methods and systems that permit improved pyrophosphate detection. Also disclosed herein are methods and systems which utilize improved pyrophosphate detection for nucleotide sequencing.

Claims

1. A method of delaying pyrophosphate detection comprising:

(a) incorporating a nucleotide or nucleotide analog into a polynucleotide, thereby generating pyrophosphate;

(b) sequestering the pyrophosphate with a pyrophosphate sequestering agent;

(c) releasing the pyrophosphate from the sequestering agent, thereby generating released pyrophosphate; and

(d) detecting the released pyrophosphate.

2. The method of claim 1, wherein the pyrophosphate is reversibly sequestered by adsorption onto the sequestering agent.

3. The method of claim 2, wherein the pyrophosphate sequestering agent comprises a cationic agent.

4. The method of claim 3, wherein the cationic agent comprises a metal, a metal salt or a metal oxide.

5. The method of claim 3, wherein the cationic agent comprises Ti or TiO₂.

6. The method of claim 2, wherein the polynucleotide is attached to a surface.

7. The method of claim 6, wherein the pyrophosphate sequestering agent is attached to the surface or to a particle.

8. The method of claim 7, further comprising a step of washing the pyrophosphate sequestering agent.

9. The method of claim 8, wherein the washing step comprises electric-field assisted removal of pyrophosphate from the pyrophosphate sequestering agent.

10. The method of claim 1, wherein the releasing of the pyrophosphate from the sequestering agent comprises providing a release reagent to the sequestering agent.

11. The method of claim 10, wherein the release reagent comprises an anionic agent.

12. The method of claim 10, wherein the release reagent comprises phosphate, bisphosphonate, or ATP sulfurylase.

13. The method of claim 1, wherein the detecting of the pyrophosphate comprises providing a pyrophosphate detecting agent.

14. The method of claim 13, wherein the polynucleotide is attached to a surface.

15. The method of claim 14, wherein the pyrophosphate sequestering agent is attached to the surface or to a particle.

16. The method of claim 15, wherein said pyrophosphate detecting agent is attached to the surface or to a particle.

17. The method of claim 13, wherein said pyrophosphate detecting agent comprises luciferase or ATP sulfurylase.

18. The method of claim 1, wherein the polynucleotide is attached to a surface.

19. The method of claim 1, further comprising repeating steps (a) through (d).

20. The method of claim 19, further comprising determining the nucleotide sequence of the polynucleotide from a sequencing-by-synthesis process that comprises the repeating of steps (a) through (d).