Probe for Nucleic Acid Sequencing and Methods of Use

• US 20080299565A1
• Filed: 12/12/2006
• Published: 12/04/2008
• Est. Priority Date: 12/12/2005
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Needle, Danielle, Lyakhov, Ilya G., Schneider, Thomas D.
• Granted Patent: US 7,871,777 B2
• Time in Patent Office: Days
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/30 Phosphoric diester hydrolysing, i.e. nuclease
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A nanoprobe for sequencing of nucleic acid molecules is provided, as well as methods for using the nanoprobe. In particular examples, the probe includes a polymerizing agent and one or more molecular linkers that carry a chemical moiety capable of reversibly binding to the template strand of a nucleic acid molecule, without being detached from the linker, by specifically binding with a complementary nucleotide in the target nucleic acid molecule. The reversible binding of the chemical moiety on the linker with a complementary nucleotide in the target nucleic acid molecule is indicated by emission of a characteristic signal that indicates pairing of the chemical moiety on the linker with its complementary nucleotide. An example of such a chemical moiety is a nonhydrolyzable nucleotide analog. In particular examples, the polymerizing agent and the chemical moiety are associated with a tag, such as a donor fluorophore and acceptor fluorophore characteristic of the particular type of chemical moiety.

Claims

1. A probe for sequencing a nucleic acid molecule, comprising:

a polymerizing agent having an active site capable of binding to a target nucleic acid molecule and promoting synthesis of a complementary nucleic acid molecule that elongates as complementary nucleotides are incorporated into the complementary nucleic acid molecule; and

one or more molecular linkers spaced apart on the polymerizing agent, wherein the one or more of the linkers carry a nucleotide analog that is capable of reversibly binding to the target nucleic acid molecule, without being detached from the linker, by specifically binding with a complementary nucleotide in the target nucleic acid molecule, wherein specific binding of the nucleotide analog on the linker with a complementary nucleotide in the target nucleic acid molecule is indicated by emission of a characteristic signal that indicates pairing of the nucleotide analog on the linker with its complementary nucleotide.

3. The probe of claim 1, wherein the nucleotide analog comprises a non-hydrolyzable nucleotide analog.

4. The probe of claim 3, wherein the non-hydrolyzable nucleotide analog comprises a non-hydrolyzable triphosphate nucleotide analog.

6. The probe of claim 1, where the nucleotide analog is a mononucleotide.

8. The probe of claim 1, wherein the one or more molecular linkers comprises at least four independent linkers, each of which carries a different nucleotide analog capable of specifically binding with a different nucleotide in the target nucleic acid molecule.

9. The probe of claim 1, wherein the one or more molecular linkers form a branch structure, wherein each branch carries a different nucleotide analog capable of specifically binding with a different nucleotide in the target nucleic acid molecule.

10. The probe of claim 9, wherein the branch structure comprises at least four branches, wherein each branch carries a different nucleotide analog capable of specifically binding with a different nucleotide in the target nucleic acid molecule.

11. The probe of claim 1, wherein the polymerizing agent is associated with a tag, and wherein each of the nucleotide analogs is associated with a tag that identifies a particular nucleotide analog carried by the linker, wherein interaction of the tag associated with the polymerizing agent with the tag associated with the nucleotide analog induces emission of the characteristic signal that indicates pairing of the nucleotide analog on the linker with its complementary nucleotide in the target nucleic acid molecule.

12. The probe of claim 11, wherein the tag associated with the polymerizing agent forms a donor-acceptor pair with the tag associated with each nucleotide analog, whereby interaction of the donor-acceptor pair stimulates emission of the characteristic signal.

15. The probe of claim 11, wherein each of the tags that identifies a particular nucleotide analog carried by the linker, comprises one or more fluorophores that emits a unique emission signal.

16. The probe of claim 1, wherein the one or more molecular linkers comprise four molecular linkers, each of which carries a different nucleotide analog capable of reversibly binding to the template strand of the target nucleic acid molecule without being detached from the linker, and an acceptor tag associated with each nucleotide analog wherein the acceptor tag identifies the particular nucleotide analog carried by the linker, and the polymerizing agent is associated with a donor tag, wherein reversible binding of the nucleotide analog to the target nucleic acid molecule brings the donor and acceptor tag into sufficient proximity to induce emission of a characteristic signal of the acceptor tag that indicates the identity of the nucleotide analog carried by the linker.

17. The probe of claim 1, wherein the one or more molecular linkers are spaced around the polymerizing agent a sufficient distance to inhibit entanglement of the molecular linkers, and the molecular linkers are of sufficient length to reach the active site of the polymerizing agent.

21. The probe of claim 1, wherein at least a portion of the molecular linker is of a sufficient rigidity to reduce interaction of the polymerizing agent and the nucleotide analog in the absence of the target nucleic acid molecule.

24. The probe of claim 1, wherein the molecular linker comprises a double-stranded DNA (dsDNA) of at least 10 nucleotides.

26. The probe of claim 1, wherein the molecular linker comprises polyethylene glycol (PEG).

31. The probe of claim 1 where the polymerizing agent comprises a DNA polymerase, RNA polymerase, ribosome, or reverse transcriptase.

37. A method of determining a nucleic acid sequence of a target nucleic acid molecule, comprising:

exposing the target nucleic acid molecule to the probe of claim 1 in the presence of an oligonucleotide primer and a mixture of hydrolyzable nucleotides that are capable of being incorporated into an elongating nucleic acid molecule by hybridizing with a complementary nucleotide in the target nucleic acid molecule, and replacing the nucleotide analog that reversibly binds to the template nucleic acid molecule;

detecting emission of a sequence of signals comprising emission of a plurality of the characteristic signals that indicates pairing of the nucleotide analog on the molecular linker with its complementary nucleotide.

38. The method of claim 37, wherein the polymerizing agent is associated with a tag, and each of the chemical moieties is also associated with a tag that identifies a particular nucleotide analog carried by the linker, wherein interaction of the tag associated with the polymerizing agent with the tag associated with the nucleotide analog induces emission of the characteristic signal that indicates pairing of the nucleotide analog on the linker with its complementary nucleotide.

39. The method of claim 38, wherein the tag associated with the polymerizing agent comprises a donor fluorophore and the tag that identifies a particular nucleotide analog comprises one or more acceptor fluorophores, wherein interaction of the polymerizing agent and the nucleotide analog that specifically binds to the complementary nucleotide in the target nucleic acid molecule brings the acceptor fluorophore into a proximity with a donor fluorophore to permit excitation of the acceptor fluorophore by the donor fluorophore.

40. The method of claim 39, wherein detecting the signal comprises detecting a fluorescent signal emitted from the acceptor fluorophore or comprises detecting a reduction in fluorescent signal emitted from the donor fluorophore.

41. The method of claim 37, wherein the emission of a sequence of signals is converted into a nucleic acid sequence.

43. The method of claim 39, further comprising exciting the donor fluorophore to emit a signal which excites the one or more acceptor fluorophores to emit the characteristic signal that indicates pairing of the nucleotide analog on the linker with its complementary nucleotide.

51. The method of claim 37, wherein the probe is fixed to a substrate.

54. The method of claim 37, wherein the target nucleic acid molecule or the oligonucleotide primer is fixed to a substrate.

55. The method of claim 37, wherein the method comprises performing a plurality of sequencing reactions substantially simultaneously, and detecting the sequence of signals from the plurality of sequencing reactions.

56. The method of claim 55, wherein a plurality of polymerizing agents, target nucleic acid molecules, or oligonucleotide primers are fixed directly or indirectly to the substrate in a predetermined pattern, and detecting the sequence of signals further comprises correlating the signal with a nucleic acid molecule corresponding to a predetermined position within that pattern.

62. The method of claim 37, wherein the target nucleic acid molecule is present in a biological sample obtained from a subject.

63. The method of claim 37, wherein the target nucleic acid molecule is present in a cell, and exposing the template nucleic acid molecule to an oligonucleotide primer and the probe comprises introduction of the oligonucleotide primer and the probe into the cell.

65. The method of claim 37, wherein the target nucleic acid strand comprises one or more mutations associated with disease.

66. The probe of claim 1, further comprising a primer that specifically hybridizes to the target nucleic acid sequence under high stringency conditions, wherein the primer is attached to the polymerizing agent via a molecular linker.

68. The probe of claim 11, wherein the tags associated with the nucleotide analogs are a coded fluorophore set that permits the detection and correction of errors.

2. (canceled)

5. (canceled)

7. (canceled)

13-14. -14. (canceled)

18-20. -20. (canceled)

22-23. -23. (canceled)

25. (canceled)

27-30. -30. (canceled)

32-35. -35. (canceled)

36. A polymerizing agent comprising:

an active site capable of binding to a target nucleic acid molecule and promoting synthesis of a complementary nucleic acid molecule that elongates as complementary nucleotides are incorporated into the complementary nucleic acid molecule;

one or more molecular linkers spaced apart on the polymerizing agent to inhibit entanglement, wherein each linker carries a different nonhydrolyzable nucleotide analog that is capable of reversibly binding to the template strand of a nucleic acid molecule, without being detached from the linker, by specifically binding with a complementary nucleotide in the target nucleic acid molecule;

a tag associated with each nonhydrolyzable nucleotide analog that identifies the nonhydrolyzable nucleotide analog carried by the linker that is capable of reversibly binding to the template strand of a nucleic acid molecule; and

a tag associated with the polymerase that interacts with the tag associated with the nonhydrolyzable nucleotide analog to emit a characteristic signal that identifies the nonhydrolyzable nucleotide analog carried by the linker.

42. (canceled)

44-50. -50. (canceled)

52-53. -53. (canceled)

57-61. -61. (canceled)

64. (canceled)

67. (canceled)