Methods and compositions to detect nucleic acids in a biological sample

• US 20060068417A1
• Filed: 06/29/2005
• Published: 03/30/2006
• 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.
• Granted Patent: US 8,034,554 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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:

providing a sample containing a target nucleic acid,

mixing the sample with a nucleic acid capture probe that forms under hybridizing conditions a partially double-stranded hairpin structure made up of

an internal target-complementary sequence, flanked by

a capture region, and

a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure in which the target-complementary region forms a substantially single-stranded loop portion,

specifically hybridizing the target-complementary sequence of the capture probe to a target sequence in the target nucleic acid,

binding the 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 target nucleic acid, the capture probe, and the immobilized probe attached to the capture support,

separating the capture hybrid attached to the capture support from sample components,

releasing the target nucleic acid from the capture hybrid,

then specifically hybridizing a detection probe to the target nucleic to form a detection hybrid, and

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 located near the 3′

end of the capture probe and the terminal region is located near the 5′

end of the capture probe.

3. The method of claim 1, wherein the capture region is located near the 5′

end of the capture probe and the terminal region is located near the 3′

end of the capture probe.

4. The method of claim 1, wherein binding the capture region to the immobilized probe hybridizes complementary sequences of the capture region and the immobilized probe.

5. 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.

6. The method of claim 1, wherein releasing the target nucleic acid from the capture hybrid further releases the capture probe from the immobilized probe.

7. 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.

8. The method of claim 1, wherein the detection probe hybridizes specifically to a target sequence that differs from or overlaps the target sequence that hybridizes to the target-complementary sequence of the capture probe.

9. The method of claim 1, wherein, in the detecting step, the signal is produced in a homogeneous reaction.

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

providing a sample containing a target nucleic acid,

mixing the sample with a capture probe that is at least a partially double-stranded structure made up of a first strand and a second strand of nucleic acid, wherein

the first strand includes a target-complementary region and a capture region, and

the second strand contains a sequence complementary to a sequence of the first strand,

specifically hybridizing the target-complementary region of the capture probe to a target sequence in the target nucleic acid,

binding the capture region to an immobilized probe attached to a capture support, thereby forming a capture hybrid made up of the target nucleic acid, the first strand of the capture probe, and the immobilized probe attached to the capture support,

separating the capture hybrid attached to the capture support from other sample components,

releasing the target nucleic acid from the capture hybrid,

then specifically hybridizing a detection probe to the target nucleic acid to form a detection hybrid, and

detecting a signal produced from the detection hybrid, thereby indicating the presence of the target nucleic acid in the sample.

11. The method of claim 10, wherein the first strand contains a 5′

capture region covalently linked to a 3′

target-complementary region, and the second strand contains a 3′

sequence complementary to the capture region of the first strand, thereby forming a partially double-stranded structure when the capture region of the first strand hybridizes to the complementary 3′

sequence of the second strand.

12. The method of claim 10, wherein the first strand contains a 5′

target-complementary region covalently linked to a 3′

capture region, and the second strand contains a 5′

sequence complementary to the 3′

capture region of the first strand, thereby forming a partially double-stranded structure when the capture region of the first strand hybridizes to the complementary 5′

sequence of the second strand.

13. A nucleic acid capture probe that forms at least a partially double-stranded structure under hybridizing conditions, comprising a target-complementary sequence and a capture region that binds to an immobilized probe by using members of a specific binding pair.

14. The capture probe of claim 13, wherein the partially double-stranded structure is a hairpin structure made up of a contiguous linear sequence that includes an internal target-complementary sequence, flanked by the capture region and a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure and the target-complementary region forms a substantially single-stranded loop portion of the hairpin structure.

15. The capture probe of claim 13, where the structure is made of up of a first strand that includes the target-complementary region and the capture region, and a separate second strand that includes a sequence complementary to a sequence of the first strand such that hybridization of the complementary sequences of the first strand and second strand produce at least a partially double-stranded structure.

16. A kit that includes the capture probe of claim 14 and a detection probe that hybridizes specifically to a target nucleic acid that contains a sequence that hybridizes to the target-complementary sequence of the capture probe.

17. The kit of claim 16, wherein the detection probe hybridizes specifically to the same target nucleic acid sequence that hybridizes to the target-complementary sequence of the capture probe.

18. The kit of claim 16 that further includes an immobilized probe attached to a capture support, in which the immobilized probe includes a member of a specific binding pair that binds specifically to the capture probe.

19. The kit of claim 18, wherein the specific binding pair members are complementary sequences that hybridize the capture probe to the immobilized probe under hybridizing conditions.

20. A kit that includes the capture probe of claim 15 and a detection probe that hybridizes specifically to a target nucleic acid that contains a sequence that hybridizes to the target-complementary sequence of the capture probe.