Amplification and detection of ribonucleic acids

• US 9,624,534 B2
• Filed: 07/13/2016
• Issued: 04/18/2017
• 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:
  US15/209,090
• Publication Number:
  US 20160312277A1
• Time in Patent Office: 279 Days
• Field of Search: 435 911, 435 612, 435 912
• 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) fragmenting a plurality of RNA molecules to generate a plurality of RNA fragments;

(b) forming a single ligation reaction mixture containing

(i) the plurality of RNA fragments, and

(ii) a plurality of a first double-stranded nucleic acid adaptor, and

(iii) a plurality of a second double-stranded nucleic acid adaptor, and

(iv) 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 at least one of the RNA fragments; and

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

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 plurality of RNA molecules comprise a small non-coding RNA or mRNA or polyA RNA.

4. The method of claim 1, wherein the fragmenting the plurality of RNA molecules is performed with RNase III.

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

6. The method of claim 5, wherein the RNA ligase comprises bacteriophage T4 RNA ligase 2 (Rnl2).

7. 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.

8. The method of claim 1, wherein at least one of the first double-stranded nucleic acid adaptors of the plurality, or at least one of the second double-stranded nucleic acid adaptors of the plurality, comprises a unique identification sequence.

9. 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.

10. 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 a portion of the at least one of the RNA fragments.

11. 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, comprises adenosine, guanosine, cytidine, and thymidine.

12. 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.

13. 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.

14. 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.

15. 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.

16. The method of claim 14, wherein the at least one forward amplification primer or the at least one reverse amplification primer includes a unique identification sequence.

17. The method of claim 14, wherein the at least one forward amplification primer or the at least one reverse amplification primer includes a promoter sequence selected from the group consisting of a T3 RNA polymerase promoter sequence, a T7 RNA polymerase promoter sequence and an SP6 RNA polymerase promoter sequence.

18. The method of claim 2, wherein the detecting of step (c) comprises sequencing the plurality of amplification products.

19. The method of claim 18, wherein the sequencing comprises sequencing with dideoxy nucleotides.

20. The method of claim 18, wherein the sequencing comprises massively parallel sequencing.