Detection of nucleic acid sequences by invader-directed cleavage

• US 5,846,717 A
• Filed: 01/24/1996
• Issued: 12/08/1998
• 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: Lyamichev, Victor, Brow, Mary Ann D., Olive, David Michael, Hall, Jeff Steven Grotelueschen, Prudent, James Robert
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SHOEMAKER, DEBRA GUNNERSEN
• Application Number:
  US08/599,491
• Time in Patent Office: 1,049 Days
• Field of Search: 435/6, 435/91.2, 435/91.5, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 530/350, 530/388.21, 935/77, 935/78, 935/91.1, 935/91.58
• US Class Current: 435/6.18
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales

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 5'"'"' 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.

Claims

1. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:

a) providing;

i) a cleavage means,

ii) a source of target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region;

iii) first and second oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5'"'"' portion of said second oligontcleotide contains sequence complementary to said first region of said target nucleic acid;

b) mixing said cleavage means, said target nucleic acid, said first oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3'"'"' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5'"'"' portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5'"'"' and 3'"'"' portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 3'"'"' portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and

c) detecting said non-target cleavage products.

2. The method of claim 1 wherein said reaction temperature is between approximately 50 and 75 degrees centigrade.

3. The method of claim 1 wherein said target nucleic acid comprises single-stranded DNA.

4. The method of claim 1 wherein said target nucleic acid comprises double-stranded DNA and prior to step c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.

5. The method of claim 4 wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.

6. The method of claim 1 wherein said target nucleic acid comprises RNA and wherein said first and second oligonucleotides comprise DNA.

7. The method of claim 1 wherein said cleavage means comprises a thermostable 5'"'"' nuclease.

8. The method of claim 7 wherein a portion of the amino acid sequence of said nuclease is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.

9. The method of claim 8 wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.

10. The method of claim 9 wherein said nuclease is encoded by a DNA sequence selected from the group consisting of SEQ ID NOS:

1-3, 9, 10, 12, 21, 30 and 31.

11. The method of claim 1 wherein said first oligonucleotide is completely complementary to said target nucleic acid and wherein said second oligonucleotide is completely complementary to said target nucleic acid.

12. The method of claim 1 wherein said 3'"'"' portion of said first oligonucleotide is partially complementary to said target nucleic acid.

13. The method of claim 1 wherein said 5'"'"' portion of said second oligonucleotide is partially complementary to said target nucleic acid.

14. The method of claim 1 wherein said detection of said non-target cleavage products comprises electrophoretic separation of the products of said reaction followed by visualization of said separated non-target cleavage products.

15. The method of claim 1 wherein said source of target nucleic acid comprises a sample containing genomic DNA.

16. The method of claim 15 wherein said sample is selected from the group comprising blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.

17. The method of claim 1 wherein said reaction conditions comprise providing a source of divalent cations.

18. The method of claim 17 wherein said divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ ions.

19. A method for detecting the presence of a target nucleic acid in a sample by generating non-target cleavage products, comprising:

a) providing;

i) a cleavage means;

ii) a sample suspected of containing a target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region;

iii) first and second oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5'"'"' portion of said second oligonucleotide contains sequence complementary to said first region of said target nucleic acid;

b) mixing said cleavage means and said first and said second oligonucleotides to create a reaction mixture under reaction conditions wherein said target nucleic acid and said first and second oligonucleotides form one or more cleavage stuctures, and wherein the combined melting temperature of said complementary regions within said 5'"'"' and 3'"'"' portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 3'"'"' portion of said first oligonucleotide, and wherein said cleavage means cleaves said cleavage structures resulting in the cleavage of said first oligonucleotide; and

c) distinguishing said cleaved first oligonucleotide from said uncleaved first oligonucleotide, said second oligonucleotide and said target nucleic acid.

20. The method of claim 19 wherein said distinguishing comprises electrophoresis of said reaction mixture after said cleavage has occurred to separate said cleaved first oligonucleotide from said uncleaved first oligonucleotide, said second oligonucleotide and said target nucleic acid followed by visualization of said separated cleaved first oligonucleotide.

21. The method of claim 20 wherein said first oligonucleotide contains a fluorescent label and said visualization consists of detecting said label using a fluorescence imager.

22. The method of claim 19 wherein said first oligonucleotide is present in excess relative to said target nucleic acid.

23. The method of claim 19 further comprising measuring the amount of cleaved first oligonucleotide such that the amount of said target nucleic acid present in said sample can be determined.

24. The method of claim 19 wherein said conditions of step b) comprise the use of a cleavage reaction temperature which is less than the melting temperature of said first oligonucleotide and greater than the melting temperature said 3'"'"' portion of said first oligonucleotide.

25. The method of claim 19 wherein said cleavage means comprises a thermostable 5'"'"' nuclease.

26. The method of claim 25 wherein a portion of the amino acid sequence of said nuclease is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.

27. The method of claim 26 wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.

28. The method of claim 27 wherein said nuclease is encoded by a DNA sequence selected from the group consisting of SEQ ID NOS:

1-3, 9, 10, 12, 21, 30 and 31.

29. A method of detecting sequence variation in a plurality of nucleic acid target sequences wherein said target nucleic acid sequences differ in sequence, comprising:

a) providing;

i) a cleavage means;

ii) a sample suspected of containing a first target nucleic acid and a second target nucleic acid, wherein said first and said second target nucleic acid have a first region, a second region and a third region;

wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region and wherein the sequence of said first and second target nucleic acids differ from one another by at least one nucleotide within their respective third regions;

iii) first and second oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said first oligonucleotide contains a sequence complementary to said third region of said first and said second target nucleic acid and wherein said 5'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said first and said second target nucleic acids, and wherein said 5'"'"' portion of said second oligonucleotide contains sequence complementary to said first region of said first and said second target nucleic acid;

iv) a third oligonucleotide having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said third oligonucleotide contains a sequence complemetary to said third region of said second target nucleic acid and wherein said 5'"'"' portion of said third oligonucleotide contains a sequence complementary to said second region of said first and said second target nucleic acid;

b) mixing said cleavage means, said first and said second target nucleic acids, said first oligonucleotide, said second oligonucleotide and said third oligonucleotide to create a reaction mixture under reaction conditions such that said first and second target nucleic acids and said first, said second and said third oligonucleotides form one or more cleavage structures, and wherein the combined melting temperature of said complementary regions within said 5'"'"' and 3'"'"' portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 3'"'"' portion of said first oligonucleotide, and said cleavage means cleaves said cleavage structures resulting in the cleavage of one or more of said first and said second oligonucleotides;

c) distinguishing said cleaved first and said second oligonucleotides from said uncleaved first and second oligonucleotides, said third oligonucleotide and said first and said second target nucleic acids.

30. The method of claim 29 wherein said first oligonucleotide contains a first label and said second oligonucleotide contains a second label.

31. The method of claim 30 wherein said distinguishing comprises separation of said cleaved first oligonucleotide from said uncleaved first oligonucleotide, said second oligonucleotide and said target nucleic acid and further comprising the step d) of detecting said separated cleaved and uncleaved products to permit a determination of the presence and relative abundance of said first and said second target nucleic acids in said sample.

32. The method of claim 29 wherein said conditions of step b) comprise the use of a cleavage reaction temperature which is less than the melting temperature of said first and said second oligonucleotides and greater than the melting temperature of said 3'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotide.