Use of single-stranded nucleic acid binding proteins in sequencing

• US 20060024678A1
• Filed: 07/28/2004
• Published: 02/02/2006
• Est. Priority Date: 07/28/2004
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Philip Richard
• Application Number:
  US10/900,711
• Publication Number:
  US 20060024678A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins

Abstract

The invention provides methods for stabilizing a nucleic acid sequencing reaction. Generally, methods of the invention include exposing a target nucleic acid to a single-stranded nucleic acid binding protein and performing a sequencing reaction.

Claims

1. A method for stabilizing a nucleic acid sequencing reaction, the method comprising the steps of:

exposing a mixture comprising a template, a polymerase, a primer, and at least one nucleotide to a single-stranded nucleic acid binding protein;

wherein said single-stranded nucleic acid binding protein binds to said template.

2. The method of claim 1, wherein said single-stranded nucleic acid binding protein increases the fidelity of said polymerase upon binding of said protein to said template.

3. The method of claim 1, wherein said nucleotide comprises a detectable label.

4. The method of claim 2, further comprising the detection of said nucleotide into said primer.

5. The method of claim 1, wherein said template is attached to a substrate such that it is individually optically resolvable.

6. The method of claim 1, wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.

7. The method of claim 1, wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.

8. The method of claim 6 or 7, wherein said first and second labels are fluorophores.

9. The method of claim 8, wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.

10. The method of claim 1, wherein said nucleotide is a nucleotide analog.

11. The method of claim 10, wherein said nucleotide analog is a chain terminating analog.

12. A method for sequencing a polynucleotide, the method comprising the steps of:

(a) stabilizing a nucleic acid template/primer complex with a single-stranded nucleic acid binding protein;

(b) exposing said complex to a polymerase and at least one nucleotide capable of extending said primer;

(c) determining whether said nucleotide extends said primer;

(d) repeating said exposing and determining steps; and

(e) compiling a sequence of said polynucleotide based upon an order of nucleotides added to said primer.

13. The method of claim 12, wherein said template/primer complex is attached to a substrate such that it is individually optically resolvable.

14. The method of claim 12, further comprising the step of removing unincorporated nucleotide.

15. The method of claim 12, wherein said nucleotide comprises a detectable label.

16. The method of claim 15, further comprising the step of rendering said label undetectable subsequent to determining step.

17. The method of claim 15, wherein said nucleotide is labeled with a fluorophore.

18. The method of claim 15, wherein said determining step comprises optically detecting incorporation of said nucleotide.

19. The method of claim 12, wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.

20. The method of claim 12, wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.

21. The method of claim 19 or 20, wherein said first and second labels are fluorophores.

22. The method of claim 21, wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.

23. The method of claim 12, wherein said nucleotide is a nucleotide analog.

24. The method of claim 23, wherein said nucleotide analog is a chain terminating analog.

25. A method for sequencing a nucleic acid template, the method comprising the steps of:

(a) exposing a nucleic acid template to a labeled nucleotide, a polymerase and a single-stranded nucleic acid binding protein under conditions that allow incorporation of said nucleotide into a primer attached to said template, wherein said single-stranded nucleic acid binding protein increases fidelity of said polymerase upon binding of said protein to said template;

(b) detecting incorporation of said nucleotide into said primer;

(c) repeating steps (a) and (b) at least once; and

(d) compiling a sequence of said template based upon an order of incorporated nucleotides.

26. The method of claim 25, wherein said template is attached to a substrate such that it is individually optically resolvable.

27. The method of claim 25, further comprising the step of removing unincorporated nucleotide.

28. The method of claim 25, further comprising the step of rendering said label undetectable subsequent to said detecting step.

29. The method of claim 25, wherein said nucleotide is labeled with a fluorophore.

30. The method of claim 29, wherein said detecting step comprises optically detecting incorporation of said nucleotide.

31. The method of claim 25, wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.

32. The method of claim 25, wherein said nucleotide is labeled with a first label and wherein said polymerase comprises a second label.

33. The method of claim 31 or 32, wherein said first and second labels are fluorophores.

34. The method of claim 33, wherein said detecting step comprises detecting coincident fluorescence emission of said labeled nucleotide and said second label.

35. The method of claim 25, wherein said nucleotide is a nucleotide analog.

36. The method of claim 35, wherein said nucleotide analog is a chain terminating analog.

39. The method of claim 25 or 37, wherein said single-stranded nucleic acid binding protein is attached to a substrate, and wherein said single-stranded nucleic acid binding protein binds said template, thereby attaching said template to said substrate.

37. A method for sequencing a nucleic acid template, the method comprising the steps of:

(a) exposing a nucleic acid template to a nucleotide, a polymerase and a single-stranded nucleic acid binding protein under conditions that allow incorporation of said nucleotide into a primer attached to said template, said nucleotide comprising a first label, and said single-stranded nucleic acid binding protein comprising a second label,

wherein one of said first and second labels comprises a donor fluorophore and the other of said labels comprises an acceptor fluorophore; and

wherein, upon binding of said single-stranded nucleic acid binding protein to said template and incorporation of said nucleotide analogue into said primer, said acceptor fluorophore is optically detectable;

(b) detecting said acceptor fluorophore, thereby to detect incorporation of said nucleotide into said primer;

(c) repeating steps (a) and (b) at least once; and

(d) compiling a sequence of said template based upon an order of incorporated nucleotides.

38. The method of claim 37, wherein said template is attached to a substrate such that it is individually optically resolvable.

40. The method of claim 37, further comprising the step of removing unincorporated nucleotide.

41. The method of claim 37, further comprising the step of rendering said acceptor fluorophore undetectable subsequent to said detecting step.

42. A method for sequencing a nucleic acid template, the method comprising the steps of:

(a) exposing a nucleic acid template to a labeled nucleotide and a polymerase under conditions that allow incorporation of said nucleotide into a primer attached to said template, said template being attached to a substrate-bound single-stranded nucleic acid binding protein such that said template is individually optically resolvable;

(b) detecting incorporation of said nucleotide into said primer;

(c) repeating steps (a) and (b) at least once; and

(d) compiling a sequence of said template based upon an order of incorporated nucleotides.

43. The method of claim 42, wherein said single-stranded nucleic acid binding protein increases fidelity of said polymerase upon binding of said protein to said template.

44. The method of claim 42, wherein said nucleotide is labeled with a first label and wherein said single-stranded nucleic acid binding protein comprises a second label.

45. The method of claim 44, wherein said first and second labels are fluorophores.