Systems and methods for invasive cleavage reaction on dendrimers

• US 6,692,917 B2
• Filed: 08/27/2001
• Issued: 02/17/2004
• Est. Priority Date: 11/29/1996
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Neri, Bruce P., Hall, Jeff G., Smith, Lloyd M., Lyamichev, Victor
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/940,244
• Publication Number:
  US 20030044796A1
• Time in Patent Office: 904 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 435/6.1
• 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 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on dendrimers and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, a 5′ nuclease activity from any 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.

Claims

1. A system comprising oligonucleotides configured for hybridizing to a target nucleic acid to form an invasive cleavage structure and a dendrimer;

wherein one or more of said oligonucleotides is attached to said dendrimer.

2. The system of claim 1, wherein said oligonucleotides are attached to said dendrimer through a covalent bond.

3. The system of claim 1, wherein said oligonucleotides are attached to said dendrimer through hybridization.

4. The system of claim 1, further comprising an agent for detecting the presence of said invasive cleavage structure.

5. The system of claim 4, wherein said agent comprises a cleavage agent.

6. The system of claim 4, wherein said cleavage agent comprises a structure-specific nuclease.

7. The system of claim 6, wherein said agent comprises a 5′

nuclease.

8. The system of claim 7, wherein said 5′

nuclease comprises a FEN-1 endonuclease.

9. The system of claim 7, wherein said 5′

nuclease comprises a polymerase.

10. The system of claim 1, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′

portion and a 5′

portion, said 5′

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

11. The system of claim 10, wherein one or more of said first oligonucleotides is attached to said dendrimer.

12. The system of claim 10, wherein one or more of said second oligonucleotides is attached to said dendrimer.

13. The system of claim 10, wherein one or more of said first oligonucleotides and one or more of said second oligonucleotides is attached to said dendrimer.

14. The system of claim 10, wherein said first oligonucleotides comprise signal probe oligonucleotides.

15. The system of claim 10, wherein said first oligonucleotides comprise a fluorescent dye.

16. The system of claim 15, wherein said first oligonucleotides further comprise a quencher molecule.

17. The system of claim 1, further comprising a spacer molecule, wherein said one or more oligonucleotides is attached to said dendrimer through said spacer molecule.

18. The system of claim 17, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

19. The system of claim 1, wherein said target nucleic acid is attached to said dendrimer.

20. The system of claim 4, wherein said agent is attached to said dendrimer.

21. A method for characterizing a nucleic acid comprising:

a) providing;

i. a sample suspected of containing a target nucleic acid;

ii. oligonucleotides configured to hybridize to said target nucleic acid to form an invasive cleavage structure;

iii. a dendrimer, wherein one or more of said oligonucleotides is attached to said dendrimer; and

iv. an agent that detects the presence of an invasive cleavage structure; and

b) exposing said sample to said oligonucleotides and said agent.

22. The method of claim 21, wherein said exposing said sample to said oligonucleotides and said agent comprises exposing said sample to said oligonucleotides and said agent under conditions wherein an invasive cleavage structure is formed between a target nucleic acid and said oligonucleotides if said target nucleic acid is present in said sample.

23. The method of claim 22, further comprising the step of c) detecting said invasive cleavage structure.

24. The method of claim 21, wherein said agent comprises a cleavage agent.

25. The method of claim 24, wherein said cleavage agent comprises a structure-specific nuclease.

26. The method of claim 24, wherein said agent comprises a 5′

nuclease.

27. The method of claim 26, wherein said 5′

nuclease comprises a FEN-1 endonuclease.

28. The method of claim 26, wherein said 5′

nuclease comprises a polymerase.

29. The method of claim 21, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′

portion and a 5′

portion, said 5′

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

30. The method of claim 29, wherein one or more of said first oligonucleotides is attached to said dendrimer.

31. The method of claim 29, wherein one or more of said second oligonucleotides is attached to said dendrimer.

32. The method of claim 29, wherein one or more of said first oligonucleotides and one or more of said second oligonucleotides is attached to said dendrimer.

33. The method of claim 29, wherein said first oligonucleotides comprise signal probe oligonucleotides.

34. The method of claim 29, wherein said first oligonucleotides comprise a fluorescent dye.

35. The method of claim 34, wherein said first oligonucleotides further comprise a quencher molecule.

36. The method of claim 21, further providing a spacer molecule, wherein said one or more oligonucleotides is attached to said dendrimer through said spacer molecule.

37. The method of claim 36, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

38. The method of claim 21, wherein said target nucleic acid is attached to said dendrimer.