Amplification and detection of ribonucleic acids

• US 8,932,816 B2
• Filed: 08/06/2013
• Issued: 01/13/2015
• Est. Priority Date: 01/14/2008
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Kuersten, R. Scott
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/960,780
• Publication Number:
  US 20130331275A1
• Time in Patent Office: 525 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/137 Chromatographic separation

Abstract

Compositions, methods, and kits for detecting one or more species of RNA molecules are disclosed. In one embodiment, a first adaptor and a second adaptor are ligated to the RNA molecule using a polypeptide comprising double-strand specific RNA ligase activity, without an intervening purification step. The ligated product is reverse transcribed, then at least some of the ribonucleosides in the reverse transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, the sequence of at least part of at least one species of amplified product is determined and at least part of the corresponding RNA molecule is determined. In some embodiments, at least some of the amplified product species are detected, directly or indirectly, allowing the presence and/or quantity of the RNA molecule of interest to be determined.

Claims

1. A method for detecting an RNA molecule, comprising:

(a) forming a single ligation reaction mixture containing at least one RNA molecule and (i) a plurality of a first double-stranded nucleic acid adaptor and (ii) a plurality of a second double-stranded nucleic acid adaptor and (iii) an RNA ligase, wherein a first strand of the first double-stranded nucleic acid adaptors of the plurality include at least two ribonucleosides at the 3′

end, and a second strand of the first double-stranded nucleic acid adaptors of the plurality include a first single-stranded portion at the 5′

end, and wherein a first strand of the second double-stranded nucleic acid adaptors of the plurality include a terminal 5′

phosphate group, and a second strand of the second double-stranded nucleic acid adaptors of the plurality include a second single-stranded portion at the 3′

end, and wherein the first and the second single-stranded portions contain a nucleotide sequence that hybridizes to a portion of the at least one RNA molecule; and

(b) producing at least one RNA ligation product by ligating the first double-stranded adaptor to a first end of the at least one RNA molecule and ligating the second double-stranded adaptor to a second end of the at least one RNA molecule.

2. The method of claim 1 further comprising:

(a) producing a first strand cDNA product by reverse transcribing the at least one RNA ligation product;

(b) producing a plurality of amplification products by amplifying the first strand cDNA product; and

(c) detecting at least one amplification product.

3. The method of claim 1, wherein the RNA ligase comprises an Rnl2 family ligase.

4. The method of claim 3, wherein the RNA ligase comprises bacteriophage T4 RNA ligase 2 (Rnl2).

5. The method of claim 1, wherein the first single-stranded portion of the first double-stranded adaptors of the plurality and the second single-stranded portion of the second double-stranded adaptors of the plurality, comprise degenerate sequences.

6. The method of claim 1, wherein the first strand of the first double-stranded nucleic acid adaptor comprises at least fifteen ribonucleosides.

7. The method of claim 1, wherein the first double-stranded adaptor of the plurality or the second double-stranded adaptor of the plurality comprises at least one reporter group.

8. The method of claim 2, wherein the amplifying in step (b) comprises PCR amplifying with at least one forward amplification primer and at least one reverse amplification primer.

9. The method of claim 8, wherein the at least one forward amplification primer or the at least one reverse amplification primer includes a unique identification sequence.

10. The method of claim 1 , wherein the first double-stranded adaptors of the plurality, or the second double-stranded adaptors of the plurality, or the first and second double-stranded adaptors comprise an identification sequence.

11. The method of claim 1, wherein the RNA molecule is a small non-coding RNA or an RNA fragment.

12. The method of claim 1, wherein the first strand of the first double-stranded adaptor and the first strand of the second double-stranded adaptor comprise different nucleotide sequences.

13. The method of claim 1, wherein a concentration of at least one species of RNA molecule is depleted prior to forming the single ligation reaction mixture of step (a).

14. The method of claim 1, wherein the first single-stranded portions of the plurality of first double-stranded nucleic acid adaptors contain different sequences, and wherein the second single-stranded portions of the plurality of second double-stranded nucleic acid adaptors contain different sequences.

15. The method of claim 2, wherein the detecting comprises sequencing the amplification product.

16. The method of claim 1, wherein the first single-stranded portion or the second single-stranded portion, or the first single-stranded portion and the second single-stranded portion comprise a sequence-specific region to selectively hybridize with the portion of the RNA molecule.

17. The method of claim 1, wherein first single-stranded portion of the first double-stranded adaptors of the plurality and the second single-stranded portion of the second double-stranded adaptors of the plurality, comprises adenosine, guanosine, cytidine, and thymidine.

18. The method of claim 2, wherein the reverse transcribing of step (a) comprises contacting the at least one RNA ligation product with triphosphate nucleotides, and (i) an RNA-dependent DNA polymerase, (ii) a DNA polymerase having DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity, or (iii) an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase.

19. The method of claim 2, wherein the amplifying in step (b) comprises contacting the first strand cDNA product with triphosphate nucleotides, at least one forward amplification primer, at least one reverse amplification primer and a DNA-dependent DNA polymerase.

20. The method of claim 19, wherein at least one of the at least one first double-stranded adaptor or at least one second double-stranded adaptor comprises an identification sequence.

21. The method of claim 19, wherein at least one of the at least one forward amplification primer or at least one reverse amplification primer comprises a reporter group.