SEPARATION OF PYROPHOSPHATE RELEASE AND PYROPHOSPHATE DETECTION

• US 20110306521A1
• Filed: 02/24/2010
• Published: 12/15/2011
• Est. Priority Date: 02/25/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Hirschbein, Bernard, Gorpe-Yasar, Filiz
• Granted Patent: US 8,445,412 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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:

providing a pyrophosphate sequestering agent;

generating pyrophosphate in the presence of said sequestering agent, whereby said pyrophosphate is reversibly sequestered;

releasing said pyrophosphate from said sequestering agent; and

detecting said pyrophosphate.

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

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

4. The method of claim 3, wherein said cationic agent comprises an agent selected from the group consisting of a metal, a metal salt, a metal oxide, hydroxyapatite, ammonium, a substituted ammonium salt, or a resin or bead comprising ammonium or a substituted ammonium salt.

5. The method of claim 4, wherein said metal, metal salt or metal oxide comprises Ti or TiO₂.

8. The method of claim 2, wherein said pyrophosphate sequestering agent comprises particles or beads.

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

10. The method of claim 9, wherein said release reagent comprises an anionic agent.

11. The method of claim 10, wherein said anionic agent comprises an agent selected from the group consisting of oxalic acid, an oxalate salt, sulfamic acid, a sulfamate salt, ethylene diamine tetraacetic acid (EDTA), and ethylene glycol-bis-β

-amino-ethyl ether N,N,N′

,N′

-tetra-acetic acid (EGTA).

12. The method of claim 9, wherein said release reagent comprises phosphate, bisphosphonate, or ATP sulfurylase.

15. The method of claim 12, wherein said ATP sulfurylase is in solution.

16. The method of claim 1, wherein detecting said pyrophosphate comprises providing a pyrophosphate detecting agent.

17. The method of claim 16, wherein said pyrophosphate detecting agent comprises luciferase.

18. The method of claim 17, wherein said pyrophosphate detecting agent further comprises ATP sulfurylase.

19. The method of claim 17, wherein said luciferase is attached to a bead.

20. The method of claim 18, wherein said ATP sulfurylase is attached to a bead.

21. The method of claim 20, wherein said ATP sulfurylase and said luciferase are both attached to the same bead.

22. The method of claim 1, wherein generating pyrophosphate in the presence of said sequestering agent comprises incorporating a nucleotide or nucleotide analog into a polynucleotide.

23. The method of claim 22, wherein said polynucleotide is attached to a surface.

24. The method of claim 23, wherein said surface comprises a bead or a well.

27. The method of claim 1, further comprising the step of washing said sequestering agent.

28. The method of claim 27, wherein washing comprises adding a washing enzyme to said sequestering agent.

29. The method of claim 28, wherein said washing enzyme is selected from the group consisting of pyrophosphatase, apyrase, alkaline phosphatase and ATP sulfurylase.

30. The method of claim 28, further comprising adding a washing enzyme inhibitor to said sequestering agent.

31. The method of claim 30, wherein said washing enzyme inhibitor is sodium azide.

32. The method of claim 27, wherein said washing step comprises electric-field assisted removal of pyrophosphate from said sequestering agent.

6-7. -7. (canceled)

13-14. -14. (canceled)

25-26. -26. (canceled)

33. A method for sequencing a nucleic acid, said method comprising:

providing nucleotides or nucleotide analogs in the presence of a pyrophosphate sequestering agent and a pyrophosphate detecting agent;

incorporating one or more of said nucleotides or nucleotide analogs into a polynucleotide so as to extend said polynucleotide in the presence of said pyrophosphate sequestering agent, thereby generating sequestered pyrophosphate;

removing said unincorporated nucleotides or nucleotide analogs from the presence of said pyrophosphate detecting agent;

releasing said pyrophosphate from said sequestering agent in the presence of said pyrophosphate detecting agent; and

detecting released pyrophosphate, wherein released pyrophosphate indicates that one or more nucleotides or nucleotide analogs have been incorporated into said polynucleotide.

34. A method of modulating the availability of free pyrophosphate during sequencing of a nucleic acid molecule, said method comprising:

combining nucleotides or nucleotide analogs with a nucleic acid template;

incubating the nucleic acid template and said nucleotides or nucleotide analogs together with a polymerase and a pyrophosphate sequestering agent under conditions sufficient to form a polynucleotide complementary to all or a portion of the nucleic acid template, wherein pyrophosphate generated during said incubating is reversibly sequestered by the sequestering agent;

removing from said nucleic acid template the nucleotides or nucleotide analogs that have not been incorporated into the polynucleotide; and

releasing said pyrophosphate from said pyrophosphate sequestering agent by providing a release reagent.

35. An array comprising a solid support having a plurality of sites distributed thereon, wherein at least a portion of said sites comprise a template nucleic acid and a pyrophosphate sequestering agent capable of reversibly sequestering pyrophosphate.

36. A method of making an array, said method comprising:

providing a solid support having a plurality of sites distributed thereon; and

providing a template nucleic acid and a pyrophosphate sequestering agent capable of reversibly sequestering pyrophosphate to at least a portion of said sites.