Detection of nucleic acids by multiple sequential invasive cleavages 02

• US 6,458,535 B1
• Filed: 07/09/1999
• Issued: 10/01/2002
• Est. Priority Date: 01/24/1996
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Mast, Andrea L., Hall, Jeff G., Lyamichev, Victor I., Brow, Mary Ann D.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US09/350,597
• Time in Patent Office: 1,180 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 435/91.5, 435/91.53, 536/23.1, 536/24.33
• US Class Current: 435/6.18
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/109 Invader technology

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 human cytomegalovirus nucleic acid in a sample.

Claims

1. A method of detecting the cleavage of nucleic acid cleavage structures comprising:

a) providing;

i) a cleavage agent;

ii) a first target nucleic acid, said first target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;

iii) a first oligonucleotide, wherein at least a section of said first oligonucleotide is completely complementary to said first region of said first target nucleic acid;

iv) a second oligonucleotide comprising a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said first target nucleic acid;

v) a second target nucleic acid, said second target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region; and

vi) a third oligonucleotide, wherein at least a portion of said third oligonucleotide is completely complementary to said first region of said second target nucleic acid;

b) generating a first cleavage structure wherein at least a portion of said section of said first oligonucleotide is annealed to said first region of said first target nucleic acid to form a double-stranded region and wherein at least said 5′

portion of said second oligonucleotide is annealed to said second region of said first target nucleic acid and wherein cleavage of said first cleavage structure within said double-stranded region occurs via said cleavage agent thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide comprising a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said second target nucleic acid;

c) generating a second cleavage structure under conditions wherein at least said portion of said third oligonucleotide is annealed to said first region of said second target nucleic acid and wherein at least said 5′

portion of said fourth oligonucleotide is annealed to said second region of said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a cleavage fragment; and

d) detecting the cleavage of said second cleavage structure.

2. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises detecting said cleavage fragment.

3. The method of claim 1, wherein said 3′

portion of said second oligonucleotide comprises a 3′

terminal nucleotide not complementary to said first target nucleic acid.

4. The method of claim 1, wherein said 3′

portion of said fourth oligonucleotide comprises a 3′

terminal nucleotide not complementary to said second target nucleic acid.

5. The method of claim 1, wherein said 3′

portion of said second oligonucleotide comprises a 3′

terminal nucleotide not complementary to said first target nucleic acid and said fourth oligonucleotide comprises a 3′

terminal nucleotide not complementary to said second target nucleic acid.

6. The method of claim 1, wherein said 3′

portion of said second oligonucleotide consists of a single nucleotide not complementary to said first target nucleic acid.

7. The method of claim 1, wherein said 3′

portion of said fourth oligonucleotide consists of a single nucleotide not complementary to said second target nucleic acid.

8. The method of claim 1, wherein said 3′

portion of said second oligonucleotide consists of a single nucleotide not complementary to said first target nucleic acid and said fourth oligonucleotide consists of a single nucleotide not complementary to said second target nucleic acid.

9. The method of claim 1, wherein said second target nucleic acid further comprises a 5′

region, wherein said 5′

region of said second target nucleic acid is said third oligonucleotide.

10. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises detection of fluorescence.

11. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises detection of mass.

12. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises detection of fluorescence energy transfer.

13. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.

14. The method of claim 1, wherein at least one of said first oligonucleotide and said second oligonucleotide is attached to a solid support.

15. The method of claim 1, wherein said third oligonucleotide is attached to a solid support.

16. The method of claim 15, wherein said third oligonucleotide is attached to a solid support at its 3′

end.

17. The method of claim 1, wherein said cleavage agent is a structure-specific nuclease.

18. The method of claim 17, wherein said structure-specific nuclease is a thermostable structure-specific nuclease.

19. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises:

a) providing;

i) said cleavage fragment;

ii) a composition comprising two single-stranded nucleic acids annealed so as to define a single-stranded portion of a protein binding region; and

iii) a protein; and

b) exposing said cleavage fragment to said single-stranded portion of said protein binding region under conditions such that said protein binds to said protein binding region.

20. The method of claim 19, wherein said protein comprises a nucleic acid producing protein and wherein said nucleic acid producing protein binds to said protein binding region and produces nucleic acid.

21. The method of claim 20, wherein said protein binding region is a template-dependent RNA polymerase binding region.

22. The method of claim 21, wherein said template-dependent RNA polymerase binding region is a T7 RNA polymerase binding region.

23. The method of claim 1, wherein said detecting the cleavage of said second cleavage structure comprises:

a) providing;

i) said cleavage fragment;

ii) a single continuous strand of nucleic acid comprising a sequence defining a single strand of an RNA polymerase binding region;

iii) a template-dependent DNA polymerase; and

iv) a template-dependent RNA polymerase; and

b) exposing said cleavage fragment to said RNA polymerase binding region under conditions such that said cleavage fragment binds to a portion of said single strand of said RNA polymerase binding region to produce a bound cleavage fragment;

c) exposing said bound cleavage fragment to said template-dependent DNA polymerase under conditions such that a double-stranded RNA polymerase binding region is produced; and

d) exposing said double-stranded RNA polymerase binding region to said template-dependent RNA polymerase under conditions such that RNA transcripts are produced.

24. The method of claim 23 further comprising the step of e) detecting said RNA transcripts.

25. The method of claim 23, wherein said template-dependent RNA polymerase is T7 RNA polymerase.

26. A method of detecting the cleavage of nucleic acid cleavage structures comprising:

a) providing;

i) a cleavage agent;

ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and a third region, said second region downstream of and contiguous to said first region and said third region upstream of said first region;

iii) a first oligonucleotide, wherein at least a section of said first oligonucleotide is completely complementary to said first region of said first target nucleic acid;

iv) a second oligonucleotide comprising a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said first target nucleic acid;

v) a second target nucleic acid, said second target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;

vi) a third oligonucleotide, wherein at least a portion of said third oligonucleotide is completely complementary to said first region of said second target nucleic acid; and

vii) a fourth oligonucleotide complementary to said third portion of said first target nucleic acid; and

b) generating a first cleavage structure wherein said fourth oligonucleotide is annealed to said third region of said first target nucleic acid and wherein at least a portion of said section of said first oligonucleotide is annealed to said first region of said first target nucleic acid to form a double-stranded region and wherein at least said 5′

portion of said second oligonucleotide is annealed to said second region of said first target nucleic acid and wherein cleavage of said first cleavage structure occurs within said double-stranded region via said cleavage agent thereby cleaving said first oligonucleotide to generate a fifth oligonucleotide, said fifth oligonucleotide comprising a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said second target nucleic acid;

c) generating a second cleavage structure under conditions wherein at least said portion of said third oligonucleotide is annealed to said first region of said second target nucleic acid and wherein at least said 5′

portion of said fifth oligonucleotide is annealed to said second region of said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a cleavage fragment; and

d) detecting the cleavage of said second cleavage structure.

27. A method of detecting the cleavage of nucleic acid cleavage structures comprising:

a) providing;

i) a cleavage agent;

ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and a third region, said second region downstream of and contiguous to said first region and said third region upstream of said first region;

iii) a first oligonucleotide comprising a 5′

end, wherein at least a section of said first oligonucleotide is completely complementary to said first region of said first target nucleic acid and wherein said 5′

end contains a region of self-complementarity;

iv) a second oligonucleotide comprising a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said first target nucleic acid;

v) a third oligonucleotide comprising a 3′

portion complementary to said 5′

end of said first oligonucleotide; and

b) generating a first cleavage structure wherein at least a portion of said section of said first oligonucleotide is annealed to said first region of said first target nucleic acid to form a double-stranded region and wherein at least said 5′

portion of said second oligonucleotide is annealed to said second region of said first target nucleic acid and wherein cleavage of said first cleavage structure occurs within said double-stranded region via said cleavage agent thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide comprising said 5′

end of said first oligonucleotide;

c) generating a second cleavage structure under conditions wherein at least said 3′

portion of said third oligonucleotide is annealed to said fourth oligonucleotide and wherein cleavage of said second cleavage structure occurs to generate a cleavage fragment; and

d) detecting the cleavage of said second cleavage structure.