Methods and compositions to detect nucleic acids in a biological sample

• US 8,551,766 B2
• Filed: 07/28/2010
• Issued: 10/08/2013
• Est. Priority Date: 07/01/2004
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Becker, Michael M., Majlessi, Mehrdad R.
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US12/845,640
• Publication Number:
  US 20120231551A1
• Time in Patent Office: 1,168 Days
• Field of Search: 435/6.1, 435/6.11, 435/287.2, 422/430, 536/23.1, 536/24.3
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

Abstract

Kits, reaction mixtures and methods for separating a target nucleic acid from a sample by using at least one hairpin capture probe oligonucleotide that has the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleic acid sequences that can form a double stranded stem portion, one of X or Y is a capture sequence that is a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and a′ b′ c′ comprises a target-complementary sequence flanked by X and Y to thereby form a loop portion of the hairpin, thus forming a capture hybrid that is separated from other sample components before the target nucleic acid is released from the capture support and hybridized to a detection probe that hybridizes specifically to the same sequence that is at least partially hybridized by the a′ b′ c′ portion of the capture probe, thus forming a detectable detection hybrid to indicate the presence of the target nucleic acid in the sample.

Claims

1. A kit for the detection of a target nucleic acid, wherein the kit comprises:

a capture probe having the structure 5′

-X.sub.n a′

b′

c′

Y.sub.n-3′

, wherein X and Y each comprise nucleic acid sequences that can form a double stranded portion of a hairpin structure and wherein one of X or Y is a capture sequence further comprising a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and wherein a′

b′

c′

comprises a target-complementary sequence that hybridizes specifically to a target sequence in the target nucleic acid and is flanked by X and Y to thereby form a loop portion of the hairpin structure; and

a detection probe that hybridizes specifically to a target sequence that overlaps or is the same as the target sequence hybridized by the target-complementary sequence of the capture probe.

2. The kit of claim 1, wherein the detection probe hybridizes specifically to the same target sequence hybridized by the target-complementary sequence of the capture probe.

3. The kit of claim 1, wherein the detection probe comprises a target-complementary sequence that is a′

b′

c′

.

4. The kit of claim 1 that further includes an immobilized probe attached to a capture support, wherein the immobilized probe includes a second member of a specific binding pair that binds specifically to the capture probe'"'"'s first member of a specific binding pair.

5. The kit of claim 4, wherein the first and second specific binding pair members are substantially complementary sequences that hybridize the capture probe to the immobilized probe under hybridizing conditions.

6. The kit of claim 5, wherein the first member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.

7. The kit of claim 5, wherein the second member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.

8. The kit of claim 1, wherein said target complementary sequence is synthesized with 2′

-O-methyl linkages.

9. The kit of claim 1, wherein Y is the capture sequence that forms the first member of the specific binding pair.

10. The kit of claim 9, wherein the capture probe has the structure 5′

-X.sub.n a′

b′

c′

poly(A).sub.10-30.

11. A reaction mixture for the detection of a target nucleic acid, wherein the reaction mixture comprises:

a capture probe having the structure 5′

-X.sub.n a′

b′

c′

Y.sub.n-3′

, wherein X and Y each comprise nucleic acid sequences that can form a double stranded portion of a hairpin structure and wherein one of X or Y is a capture sequence further comprising a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and wherein a′

b′

c′

comprises a target-complementary sequence that hybridizes specifically to a target sequence in the target nucleic acid and is flanked by X and Y to thereby form a loop portion of the hairpin structure; and

a detection probe that hybridizes specifically to a target sequence that overlaps or is the same as the target sequence hybridized by the target-complementary sequence of the capture probe.

12. The reaction mixture of claim 11, wherein the detection probe hybridizes specifically to the same target sequence hybridized by the target-complementary sequence of the capture probe.

13. The reaction mixture of claim 11, wherein the detection probe comprises a target-complementary sequence that is a′

b′

c′

.

14. The reaction mixture of claim 11, wherein the first member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.

15. The reaction mixture of claim 11, wherein the second member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.

16. The reaction mixture of claim 11, wherein said target complementary sequence is synthesized with 2′

-O-methyl linkages.

17. The reaction mixture of claim 11, wherein Y is the capture sequence that forms the first member of the specific binding pair.

18. The reaction mixture of claim 17, wherein the capture probe has the structure 5′

-X.sub.n a′

b′

c′

poly(A).sub.10-30.

19. The reaction mixture of claim 11, wherein the detection probe is labeled with a chemiluminescent compound.

20. The reaction mixture of claim 19, wherein the chemiluminescent compound is an acridinium ester.

21. The reaction mixture of claim 11, wherein the detection probe is labeled with a fluorescent compound.

22. The reaction mixture of claim 21, wherein the detection probe is a molecular torch or a molecular beacon.

23. The reaction mixture of claim 11, wherein the reaction mixture contains the target nucleic acid, and wherein the detection probe is hybridized to the target nucleic acid.