Detection of small nucleic acids

• US 8,609,336 B2
• Filed: 02/07/2012
• Issued: 12/17/2013
• Est. Priority Date: 12/18/2002
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Dahlberg, James E., Lyamichev, Victor, Neri, Bruce P., Olson-Munoz, Marilyn, Chehak, LuAnne, Olson, Sarah M., Allawi, Hatim T
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US13/368,191
• Publication Number:
  US 20120149018A1
• Time in Patent Office: 679 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: Y10T 436/145555 Hetero-N

Abstract

The present invention relates to compositions and methods for the detection and characterization of interfering 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 quantitation of interfering RNA expression. The present invention further provides for the detection of variants and types of miRNAs and siRNAs.

Claims

1. A method for analyzing microRNA in a sample, comprising:

a) hybridizing a microRNA target to at least one DNA nucleic acid, wherein said DNA nucleic acid contains a first region that is complementary to at least a portion of said microRNA target, and a second region that is not complementary to said microRNA target, wherein said second region of said nucleic acid forms a hairpin loop to form a half dumbbell structure when said DNA nucleic acid is hybridized to said microRNA, to generate a detection structure; and

b) detecting said detection structure.

2. The method of claim 1, further comprising reacting said detection structure with at least one of a structure-specific nuclease or a DNA polymerase to form a modified detection structure, wherein said reacting comprises cleaving and/or extending at least one of said DNA nucleic acid or said microRNA in said detection structure.

3. The method of claim 1, wherein said detecting comprises quantitating said microRNA.

4. The method of claim 1, wherein said detecting comprises forming an invasive cleavage structure, and detecting the cleavage of said invasive cleavage structure.

5. The method of claim 4, wherein said detection structure comprises first and second oligonucleotides configured to form an invasive cleavage structure in combination with said microRNA, wherein formation of an invasive cleavage structure with said microRNA requires both of said first and said second oligonucleotides.

6. The method of claim 4, wherein said detection structure comprises a DNA first oligonucleotide configured to form an invasive cleavage structure in combination with said microRNA, wherein said DNA first oligonucleotide and said microRNA in combination are sufficient to form an invasive cleavage structure.

7. The method of claim 5, further comprising a second nucleic acid, wherein said second nucleic acid contains a first region that is complementary to a portion of said microRNA target that is not complementary to said first nucleic acid, and a second region that is not complementary to said microRNA target, wherein said second region of said second nucleic acid forms a hairpin loop to form a half dumbbell structure when said nucleic acid is hybridized to said microRNA.

8. The method of claim 1, wherein said detecting comprises detecting cleavage of an invasive cleavage structure.

9. The method of claim 1, wherein said sample comprises a cell lysate.

10. The method of claim 1, wherein a plurality of different microRNAs are detected.

11. The method of claim 10, wherein said plurality of microRNAs comprise a first microRNA and a second microRNA that is said first microRNA having a polymorphism.

12. The method of claim 1, further comprising detecting a second nucleic acid target.

13. The method of claim 12, wherein said second nucleic acid target is RNA.

14. The method of claim 13, wherein said second nucleic acid target is selected from the group consisting of U6 and GAPDH.

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