Compositions for detecting small RNAs

• US 8,951,730 B2
• Filed: 03/07/2013
• Issued: 02/10/2015
• Est. Priority Date: 11/25/2008
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Siva, Amara, Reynolds, Mark A.
• Primary Examiner(s): Pande, Suchira
• Application Number:
  US13/789,409
• Publication Number:
  US 20140205999A1
• Time in Patent Office: 705 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Compositions and reaction mixtures are provided for the detection of small RNA target nucleic acids, preferably miRNA target nucleic acids, wherein the compositions and reaction mixtures provide for sensitive and specific detection of the target nucleic acids. The compositions and reaction mixtures include one or more of a first amplification oligomer that is preferably an extender primer, a target capture oligomer that is preferably at least partially double stranded, a promoter primer/provider, a reverse primer that is preferably a universal primer and a detection probe. The compositions and reaction mixtures are useful for diagnostics, prognostics, monitoring the effectiveness of treatment and/or determining a treatment.

Claims

1. An amplification reaction mixture comprising:

(a) a miR-221 target capture probe that forms under hybridizing conditions at least a partially double-stranded structure that is a hairpin structure made up of a contiguous linear sequence that includes an internal target hybridizing sequence that is substantially complementary to all or a portion of SEQ ID NO;

44 and that forms a single-stranded loop portion of the hairpin structure, wherein the internal target hybridizing sequence is flanked by a capture region and by a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure, wherein the capture region further comprises a substantially homopolymeric nucleic acid sequence and the terminal region further comprises a substantially homopolymeric nucleic acid sequence that is complementary to the capture region sequence, and wherein the miR-221 target capture probe is at least 95% identical to SEQ ID NO;

24; and

(b) a first amplification oligomer configured to hybridize to a miR-221 target nucleic acid that is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, wherein the 3′

target hybridizing sequence is substantially identical to about 9 or more contiguous nucleic acids at the 5′

end of the target hybridizing sequence of the miR-221 target capture probe.

2. The amplification reaction mixture of claim 1, further comprising a miR-182 target capture probe that forms under hybridizing conditions at least a partially double-stranded structure that is a hairpin structure made up of a contiguous linear sequence that includes an internal target hybridizing sequence that is substantially complementary to all or a portion of SEQ ID NO:

41 and that forms a single-stranded loop portion of the hairpin structure, wherein the internal target hybridizing sequence is flanked by a capture region and by a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure and wherein the capture region further comprises a substantially homopolymeric nucleic acid sequence and the terminal region further comprises a substantially homopolymeric nucleic acid sequence that is complementary to the capture region sequence.

3. The amplification reaction mixture of claim 1, further comprising a first amplification oligomer configured to hybridize to a miR-182 target nucleic acid that is an extender primer comprising a 3′

target hybridizing sequence and a 5′

extension nucleic acid sequence, wherein the 3′

target hybridizing sequence is substantially identical to about 9 or more contiguous nucleic acids at the 5′

end of the target hybridizing sequence of the miR-182 target capture probe.

4. The amplification reaction mixture of claim 1, further comprising a promoter based oligomer configured to specifically hybridize to a miR-221 target nucleic acid, that comprises a target hybridizing sequence that is substantially complementary to 12 or more contiguous nucleic acids on 3′

end of the miR-221 target capture probe.

5. The amplification reaction mixture of claim 4, wherein the miR-221 promoter based oligomer is at least 95% identical to SEQ ID NO:

11.

6. The amplification reaction mixture of claim 4, wherein the miR-221 promoter based oligomer is SEQ ID NO:

11.

7. The amplification reaction mixture of claim 1, wherein the miR-221 target capture probe is SEQ ID NO:

24.

8. The amplification reaction mixture of claim 1, wherein the miR-221 extender primer is at least 95% identical to SEQ ID NO:

17.

9. The amplification reaction mixture of claim 1, wherein the miR-221 extender primer is SEQ ID NO:

17.

10. The amplification reaction mixture of claim 2, further comprising a promoter based oligomer configured to specifically hybridize to a miR-182 target nucleic acid, that comprises a target hybridizing sequence that is substantially complementary to 12 or more contiguous nucleic acids on 3′

end of the miR-182 target capture probe.

11. The amplification reaction mixture of claim 10, wherein the promoter based oligomer is at least 95% identical to SEQ ID NO:

10.

12. The amplification reaction mixture of claim 10, wherein the promoter based oligomer is SEQ ID NO:

10.

13. The amplification reaction mixture of claim 2, wherein the miR-182 target capture probe is at least 95% identical to SEQ ID NO:

23.

14. The amplification reaction mixture of claim 2, wherein the miR-182 target capture probe is SEQ ID NO:

23.

15. The amplification reaction mixture of claim 3, wherein the miR-182 extender primer is at least 95% identical to SEQ ID NO:

16.

16. The amplification reaction mixture of claim 15, wherein the miR-182 extender primer is SEQ ID NO:

16.

17. The amplification reaction mixture of claim 1, further comprising a reverse primer.

18. The amplification reaction mixture of claim 2, further comprising a universal reverse primer.

19. The amplification reaction mixture of claim 1, wherein the target capture probe comprises at least one 2′

-OMe residue in the target hybridizing sequence.

20. The amplification reaction mixture of claim 2, wherein the target capture probe comprises at least one 2′

-OMe residue in the target hybridizing sequence.