Detection of Nucleic Acids By Multiple Sequential Invasive Cleavages

• US 20080182254A1
• Filed: 10/28/2007
• Published: 07/31/2008
• Est. Priority Date: 01/24/1996
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Hall, Jeff G., Lyamichev, Victor I., Mast, Andrea L.
• Granted Patent: US 7,514,220 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

Claims

1. A kit for detecting a target sequence, comprising:

i) nucleic acid molecules capable of forming an invasive cleavage structure in the presence of a target sequence; and

ii) an agent for detecting the presence of an invasive cleavage structure; and

2. The kit of claim 1, wherein said agent comprises a cleavage agent.

3. The kit of claim 2, wherein said cleavage agent is a structure-specific 5′

-nuclease.

4. The kit of claim 3, wherein said structure-specific 5′

-nuclease comprises a thermostable structure-specific 5-nuclease.

5. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease is active at or above 30°

C.

6. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease is active at or above 40°

C.

7. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease is active at or above 50°

C.

8. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease is active at or above 60°

C.

9. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease is active at or above 65°

C.

10. The kit of claim 4, wherein said thermostable structure-specific 5′

-nuclease comprises a Flap-endonuclease.

11. The kit of claim 10, wherein said Flap-endonuclease comprises a FEN-1 endonuclease.

12. The kit of claim 1, further comprising a polymerase.

14. The kit of claim 3, wherein a portion of said structure-specific 5′

-nuclease is homologous to a portion of a thermostable DNA polymerase derived from a thermophilic organism.

15. The kit of claim 14, wherein said thermophilic organism is selected from the group consisting of Thermus aquaticus, Thermus flavus, and Thermus thermophilus.

16. The kit of claim 1, wherein said kit further comprises a solid support.

17. The kit of claim 16, wherein at least a portion of said invasive cleavage structure is attached to said solid support.

18. The kit of claim 1, further comprising a buffer solution.

19. The kit of claim 18, wherein said buffer solution comprises a source of divalent cations.

20. The kit of claim 19, wherein said divalent cation is selected from the group consisting of Mn²⁺ and Mg²⁺ ions.

21. The kit of claim 1, further comprising said target nucleic acid.

22. The kit of claim 21, wherein said target sequence comprises a first region and a second region, said second region downstream of and contiguous to said first region, and wherein said nucleic acid molecules comprise first and second nucleic acid molecules, wherein at least a portion of said first nucleic acid molecule is completely complementary to said first region of said target sequence, and wherein said second nucleic acid molecule comprises a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said target sequence.

23. The kit of claim 1, wherein said nucleic acid molecules comprise first and second nucleic acid molecules, said first nucleic acid molecule comprising a 5′

portion complementary to a first region of said target sequence, and said second nucleic acid molecule comprising a 3′

portion and a 5′

portion, said 5′

portion complementary to a second region of said target sequence downstream of and contiguous to said first region of said target sequence.

24. The kit of claim 23, further comprising a second target sequence.

28. The kit of claim 24, wherein said agent comprises a cleavage agent.

29. The kit of claim 28, wherein said cleavage agent is a structure-specific 5′

-nuclease.

30. The kit of claim 29, wherein said structure-specific 5′

-nuclease comprises a thermostable structure-specific 5-nuclease.

35. The kit of claim 1, further comprising instructions for using said kit for detecting said presence of a target sequence.

36. The kit of claim 35, further comprising a computer readable medium comprising instructions for using said kit for detecting said presence of a target sequence.

37. The kit of claim 35, further comprising computer instructions which direct a processor to analyze data derived from use of said agent for detecting the presence of an invasive cleavage structure.

13. The kit of 1, further comprising a ligase.

25. A kit for detecting a target sequence, comprising:

a) nucleic acid molecules configured to form an invasive cleavage structure on a target sequence that is cleavable by a 5′

-nuclease, wherein said invasive cleavage structure comprises;

i) said target sequence;

iii) nucleic acid molecules that form an invasive cleavage structure with a displaced 5′

nucleic acid arm in the presence of said target sequence; and

b) an agent for detecting the presence of an invasive cleavage structure.

26. The kit of claim 25, wherein said 5′

-nuclease comprises a FEN-1 endonuclease.

27. The kit of claim 26, wherein said FEN-1 endonuclease is Mja FEN-1 endonuclease.

31. The kit of claim 25, wherein said invasive cleavage structures are formed and cleaved under isothermal conditions.

32. The kit of claim 31, wherein said isothermal conditions are at or above 35°

C.

33. The kit of claim 31, wherein said isothermal conditions are at or above 45°

C.

34. The kit of claim 31, wherein said isothermal conditions are at or above 55°

C.