Detection of nucleic acids

• US 20080131890A1
• Filed: 06/01/2007
• Published: 06/05/2008
• Est. Priority Date: 12/18/2002
• Status: Active Grant

• Current Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Lyamichev, Victor, Allawi, Hatim T.
• Granted Patent: US 8,206,904 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2561/109 Invader technology

Abstract

The present invention relates to compositions and methods for the detection and characterization of small nucleic acid molecules (e.g., RNA (e.g., small RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs)) and other short nucleic acid molecules). More particularly, the present invention relates to methods for the detection and quantification of RNA expression. The present invention further provides for the detection of miRNA and siRNA variants.

Claims

1. A method for detecting an miRNA target, comprising:

a) providing;

i) an miRNA target;

ii) a first unlabled oligonucleotide that comprises a first region that is complementary to said miRNA target and a second region that is not complementary to said miRNA target;

iii) a second unlabeled oligonucleotide that comprises a first region that is complementary to a second region of said miRNA target and a second region that is not complementary to said second region of said miRNA target;

iv) a reverse transcriptase;

v) a DNA polymerase; and

vi) a probe oligonucleotide, wherein at least a portion of said probe oligonucleotide is complementary to at least a portion of said miRNA;

b) incubating components (i) through (vi) under conditions such that a detection structure forms; and

c) detecting said detection structure.

2. The method of claim 1, wherein said detecting comprises forming an invasive cleavage structure comprising said probe oligonucleotide, cleaving said invasive cleavage structure, and detecting the cleavage of said invasive cleavage structure.

3. The method of claim 1, wherein said first unlabeled oligonucleotide is used as a primer for reverse transcription.

4. The method of claim 1, wherein said first unlabeled oligonucleotide is used as an INVADER oligonucleotide in an invasive cleavage reaction.

5. The method of claim 1, wherein components (i) through (vi) are combined in the same reaction vessel prior to said incubating.

6. The method of claim 1, wherein said providing further comprises providing (vii) an enzyme capable of cleaving a detection structure, wherein said enzyme lacks polymerase activity.

7. The method of claim 6, wherein said enzyme capable of cleaving a detection structure is a FEN-1 nuclease.

8. The method of claim 1, wherein said probe oligonucleotide is unlabeled.

9. The method of claim 1, wherein said first unlabeled oligonucleotide and said reverse transcriptase reverse transcribe said miRNA target to produce an miRNA cDNA target.

10. The method of claim 9, wherein the miRNA cDNA target is amplified by said first unlabeled oligonucleotide and said second unlabeled oligonucleotide and said DNA polymerase in a polymerase chain reaction.

11. The method of claim 10, wherein the amplified miRNA cDNA target forms a detection structure in the presence of said probe oligonucleotide.

12. The method of claim 1, wherein said first unlabeled oligonucleotide comprises nucleic acid sequence such that a duplex of about 6-10 base pairs is formed between said oligonucleotide and the miRNA target.

13. The method of claim 1, wherein said second regions of either or both of said first and said second unlabeled oligonucleotide probes comprise a first portion and a second portion, wherein the first portion and the second portion within a second region can hybridize to each other to form a hairpin structure.

14. The method of claim 1, further comprising providing (viii) an oligonucleotide complementary to a region of said first unlabeled oligonucleotide probe.

15. The method of claim 1, further comprising providing (viii) an oligonucleotide complementary to a region of said second unlabeled oligonucleotide probe.

16. The method of claim 1, wherein said detecting comprises use of a labeled probe.

17. The method of claim 16, wherein said labeled probe is configured for FRET detection.

18. The method of claim 1, wherein said miRNA is selected from the group consisting of Let-7, miR-1, miR-135, miR-15, miR-16, miR125b, miR-1d, and miR124a.

19. A kit comprising:

i) a first unlabled oligonucleotide that comprises a first region that is complementary to a miRNA target and a second region that is not complementary to said miRNA target;

ii) a second unlabeled oligonucleotide that comprises a first region that is complementary to a second region of said miRNA target and a second region that is not complementary to said second region of said miRNA target;

iii) a reverse transcriptase;

iv) a DNA polymerase;

v) a probe oligonucleotide; and

vi) an enzyme capable of cleaving a detection structure.

20. The kit of claim 19, wherein said detection structure comprises an invasive cleavage structure.