Method for detection and characterization of short nucleic acids

• US 20070020644A1
• Filed: 01/26/2006
• Published: 01/25/2007
• Est. Priority Date: 01/26/2005
• Status: Abandoned Application

• Current Assignee: Benitec, Inc. (Benitec Biopharma Limited)
• Original Assignee: Benitec, Inc. (Benitec Biopharma Limited)
• Inventors: Kolykhalov, Alexander, Schroeder, A.
• Application Number:
  US11/340,830
• Publication Number:
  US 20070020644A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

A method of detecting and characterizing a target short RNA is provided.

Claims

1. A method of detecting a target short RNA, comprising:

hybridizing at least one ligation agent to the target short RNA;

ligating the at least one ligation agent and the target short RNA with a template-dependent ligase to form a ligated molecule; and

detecting the ligated molecule.

2. The method of claim 1, wherein the at least one ligation agent is a nucleic acid.

3. The method of claim 2, wherein the nucleic acid is a DNA oligonucleotide.

4. The method of claim 1, wherein a 3′

end of the ligation agent is not a substrate for a chain extension enzyme.

5. The method of claim 4, wherein the 3′

end of the at least one ligation agent is blocked by a hydrogen, 3′

phosphoglycolate, or 3′

amine.

6. The method of claim 1, wherein the target short RNA has a length of 17-50 nucleotides.

7. The method of claim 1, wherein the target short RNA is an mRNA, siRNA, or processed shRNA.

8. The method of claim 1, wherein the at least one ligation agent comprises a portion of sequence that is not complementary to the target short RNA and a portion of sequence that is complementary to the target short RNA.

9. The method of claim 1, wherein the ligated molecule is detected by a quantitative nucleic acid amplification technique.

10. The method of claim 9, wherein the ligated molecule is detected by a real-time PCR assay.

11. The method of claim 9, wherein the ligated molecule is detected by a TaqMan™

assay.

12. The method of claim 1, wherein the ligated molecule is detected by a method selected from the group consisting of sequencing assays, polymerase chain reaction assays, hybridization assays, hybridization assays employing a probe complementary to a mutation, microarray assays, bead array assays, primer extension assays, enzyme mismatch cleavage assays, branched hybridization assays, NASBA assays, molecular beacon assays, cycling probe assays, ligase chain reaction assays, invasive cleavage structure assays, ARMS assays, and sandwich hybridization assays.