Methods and compositions to detect nucleic acids in a biological sample

• US 8,460,869 B2
• Filed: 04/14/2011
• Issued: 06/11/2013
• 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:
  US13/087,282
• Publication Number:
  US 20110250599A1
• Time in Patent Office: 789 Days
• Field of Search: 435/6.1, 435/6.11, 435/91.2, 435/287.2, 536/23.1, 536/24.5, 536/25.4
• US Class Current: 435/6.1
• 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

Methods of the invention separate a target nucleic acid from a sample by using at least one capture probe oligonucleotide that contains a target-complementary region and a member of a specific binding pair that attaches the target nucleic acid to an immobilized probe on a capture support, 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 to form a detection hybrid that produces a detectable signal that indicates the presence of the target nucleic acid in the sample. Compositions for practicing the methods of the invention include a capture probe oligonucleotide made up a target-complementary region sequence and a covalently linked capture region sequence that includes a member of a specific binding pair.

Claims

1. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:

(a) providing a sample containing a target nucleic acid,

(b) mixing the sample with a soluble nucleic acid capture probe that is configured as 5′

-capture region-target complementary sequence-terminal region-3′

or as 5′

-terminal region-target complementary sequence-capture region-3′

, and that forms under hybridizing conditions, a partially double-stranded hairpin structure wherein the capture region and the terminal region bind intramolecularly to form a double stranded stem, wherein the target-complementary region forms a single-stranded loop portion of the partially double stranded hairpin structure, and wherein the capture region and the terminal region are configured to not hybridize to the target nucleic acids under stringent hybridization conditions,

(c) specifically hybridizing the target-complementary sequence of the capture probe to a target sequence in the target nucleic acid, such that the target complementary sequence and the target sequence are in a double stranded form, and wherein the capture region and the terminal region are not intramolecularly bound one to the other, such that the capture region and the terminal region are each in a single stranded form,

(d) binding the single-stranded capture region to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the capture support, the immobilized probe, the nucleic acid capture probe and the target nucleic acid,

(e) separating the capture hybrid attached to the capture support from sample components,

(f) releasing the target nucleic acid from the capture hybrid, wherein releasing the target nucleic acid from the capture hybrid further releases the capture probe from the immobilized probe and the released the capture probe reforms the partially double-stranded hairpin structure,

(g) hybridizing a detection probe to the target nucleic acid to form a detection hybrid, wherein the detection probe hybridizes specifically to a target sequence that overlaps or is the same as the target sequence that hybridizes to the target-complementary sequence of the capture probe, wherein said detection probe hybridization is performed in the presence of the capture probe, and wherein the reformation of the capture probe'"'"'s partially double-stranded hairpin structure prevents or minimizes competition between the capture probe and the detection probe, and

(h) detecting a signal produced from the detection hybrid to indicate the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein the capture region is a nucleic acid sequence that is substantially complementary to the terminal region nucleic acid sequence.

3. The method of claim 2, wherein the immobilized probe is a nucleic acid sequence that is complementary to the capture region nucleic acid sequence.

4. The method of claim 3, wherein the immobilized probe nucleic acid sequence and the terminal region nucleic acid sequence are substantially identical nucleic acid sequences.

5. The method of claim 4, wherein the terminal region sequence has one or more of a mismatch, substitution or deletion compared to the immobilized probe nucleic acid sequence.

6. The method of claim 5, wherein the capture region is from 10 nucleobases in length to 30 nucleobases in length.

7. The method of claim 1, wherein the capture region is from 10 nucleobases in length to 30 nucleobases in length.

8. The method of claim 1, wherein binding the capture region to the immobilized probe binds together non-nucleic acid members of a specific binding pair.

9. The method of claim 1, wherein the detection probe hybridizes specifically to a target sequence that is the same target sequence that hybridizes to the target-complementary sequence of the capture probe.

10. The method of claim 1, further comprising a nucleic acid amplification step.

11. The method of claim 1, wherein the target nucleic acid is a portion of a nucleic acid selected from:

a bacterial gene, an RNA encoded by a bacterial gene, a human gene, an RNA encoded by a human gene, a human mitochondrial nucleic acid, a viral gene, or an RNA encoded by a viral gene.

12. The method of claim 1, wherein the target nucleic acid is a small RNA, is an RNA that is 17-23 nucleobases in length, is a micro RNA, is a short-hairpin RNA, is a short interfering RNA or is a small nuclear RNA.

13. The method of claim 1, wherein said target-complementary sequence is synthesized with 2′

-O-methyl linkages.

14. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:

(a) providing a sample containing a target nucleic acid,

(b) mixing said sample with a nucleic acid capture probe that has the structure 5′

-X.sub.n a′

b′

c′

Y.sub.n-3′

, wherein either X is a capture region and Y is a terminal region or X is a terminal region and Y is a capture region, and wherein n is from 10 contiguous nucleobases to 30 contiguous nucleobases in length, and wherein X and Y do not hybridize to the target nucleic acid under stringent hybridization conditions, and wherein a′

b′

c′

is the target-complementary sequence, and wherein under conditions the nucleic acid capture probe forms a partially double stranded hairpin structure that has a double-stranded stem portion made from the intramolecular binding of X to Y, and has a single stranded loop portion made by the a′

b′

c′

portion,

(c) providing conditions to said mixture, said conditions designed for specifically hybridizing a′

b′

c′

to a target sequence in the target nucleic acid, and wherein X and Y are not intramolecularly bound,

(d) binding the single stranded capture sequence from step c to an immobilized probe sequence, thereby forming a capture hybrid made up of the target nucleic acid, the capture probe, and the immobilized probe attached to a capture support,

(e) separating the capture hybrid from sample components,

(f) releasing said target nucleic acid from the capture hybrid, wherein releasing the target nucleic acid from the capture hybrid further releases the capture probe from the immobilized probe and the released the capture probe reforms the partially double-stranded hairpin structure,

(g) optionally, amplifying the target nucleic acid, and

(h) detecting the presence of the target nucleic acid or the amplified target nucleic acid, wherein said detecting comprises hybridizing the target nucleic acid to a detection probe that hybridizes specifically to a target sequence that overlaps or is the same as the target sequence that hybridizes to the target-complementary sequence of the capture probe, wherein said detection probe hybridization is performed in the presence of the capture probe, and wherein the reformation of the capture probe'"'"'s partially double-stranded hairpin structure prevents or minimizes competition between the capture probe and the detection probe.

15. The method of claim 14, wherein X is substantially complementary to Y.

16. The method of claim 14, wherein the immobilized probe is substantially identical to the terminal region.

17. The method of claim 16, wherein the immobilized probe is fully complementary to the capture region.

18. The method of claim 17, wherein the terminal region sequence has one or more of a mismatch, substitution or deletion compared to the immobilized probe nucleic acid sequence.

19. The method of claim 14, wherein the terminal region sequence has one or more of a mismatch, substitution or deletion compared to the immobilized probe nucleic acid sequence.

20. The method of claim 14, wherein the target nucleic acid is an RNA made up of at least 10 or more bases linked by a backbone structure.

21. The method of claim 14, wherein the target nucleic acid is a portion of a nucleic acid selected from:

a bacterial gene, an RNA encoded by a bacterial gene, a human gene, an RNA encoded by a human gene, a human mitochondrial nucleic acid, a viral gene, or an RNA encoded by a viral gene.

22. The method of claim 14, wherein the target nucleic acid is a small RNA, is an RNA that is 17-23 nucleobases in length, is a micro RNA, is a short-hairpin RNA, is a short interfering RNA or is a small nuclear RNA.

23. The method of claim 14, wherein said target-complementary sequence is synthesized with 2′

-O-methyl linkages.

24. The method of claim 14, wherein the detection probe hybridizes specifically to a target sequence that is the same target sequence that hybridizes to the target-complementary sequence of the capture probe.