Amplification and detection of ribonucleic acid

• US 8,192,941 B2
• Filed: 07/14/2010
• Issued: 06/05/2012
• 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:
  US12/835,869
• Publication Number:
  US 20100279305A1
• Time in Patent Office: 692 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/6.12
• 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 a RNA molecule in a sample, comprising:

combining the sample with at least one first adaptor, at least one second adaptor, and a polypeptide comprising double-strand specific RNA ligase activity to form a ligation reaction composition in which the at least one first adaptor and the at least one second adaptor are ligated to the RNA molecule of the sample to form a ligated product in the same ligation reaction composition,

wherein the at least one first adaptor comprises;

a first oligonucleotide having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3′

-end, and

a second oligonucleotide comprising a nucleotide sequence substantially complementary to the first oligonucleotide and further comprising a single-stranded 5′

portion of 1 to 8 nucleotides when the first oligonucleotide and the second oligonucleotide are duplexed,

wherein the at least one second adaptor comprises;

a third oligonucleotide having a length of 10 to 60 nucleotides and comprising a 5′

phosphate group, and

a fourth oligonucleotide comprising a nucleotide sequence substantially complementary to the third oligonucleotide and further comprising a single-stranded 3′

portion of 1 to 8 nucleotides when the third oligonucleotide and the fourth oligonucleotide are duplexed,

wherein the single-stranded portions independently have a degenerate nucleotide sequence, or a sequence that is complementary to a portion of the RNA molecule,

wherein the first and third oligonucleotides have a different nucleotide sequence;

wherein the RNA molecule hybridizes with the single-stranded portion of the at least one first adaptor and the single-stranded portion of the at least one second adaptor; and

combining the ligated product with

i) a RNA-directed DNA polymerase,

ii) a DNA polymerase comprising DNA dependent DNA polymerase activity and RNA dependent DNA polymerase activity, or

iii) a RNA-directed DNA polymerase and a DNA-directed DNA polymerase,

reverse transcribing the ligated product to form a reverse transcribed product,

digesting at least some of the ribonucleosides from the reverse transcribed product with ribonuclease H to form an amplification template,

combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase when the ligated product is combined as in i), to form an amplification reaction composition,

cycling the amplification reaction composition to form at least one amplified product, and

determining the sequence of at least part of the amplified product, thereby detecting the RNA molecule.

2. The method of claim 1, wherein the polypeptide comprising double strand-specific RNA ligase activity comprises an Rnl2 family ligase.

3. The method of claim 2, wherein the polypeptide comprising double strand-specific RNA ligase activity comprises bacteriophage T4 RNA ligase 2 (Rnl2).

4. The method of claim 1, wherein the single-stranded portion of the first adaptor, the single-stranded portion of the second adaptor, or the single-stranded portion of the first adaptor and the single-stranded portion of the second adaptor, comprise degenerate sequences.

5. The method of claim 1, wherein the first oligonucleotide comprises at least fifteen ribonucleosides.

6. The method of claim 1, wherein the second adaptor comprises at least one reporter group.

7. The method of claim 1, wherein the at least one amplified product comprises an identification sequence.

8. The method of claim 7, wherein at least one of the at least one forward primer, at least one of the at least one reverse primer, or both, comprise an identification sequence.

9. The method of claim 7, wherein at least one first adaptor, at least one second adaptor, or at least one first adaptor and at least one second adaptor comprises an identification sequence.

10. The method of claim 1, wherein the RNA molecule is a small non-coding RNA.

11. The method of claim 1, wherein the sample comprises a plurality of RNA fragments and the detecting comprises determining an expression profile.

12. The method of claim 11, wherein a concentration of at least one species of RNA molecule is depleted prior to forming the ligated product.

13. The method of claim 11, wherein the sample comprises a plurality of messenger RNA (mRNA) fragments and the detecting comprises determining an expression profile.

14. The method of claim 1, wherein the detecting comprises sequencing at least part of an amplified product, wherein the sequencing comprises a massive parallel signature sequencing reaction, hybridizing at least one amplified product to a microarray, or cloning at least one amplified product into a sequencing vector.

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

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

combining the RNA molecule with at least one first adaptor, at least one second adaptor, and a double-strand specific RNA ligase to form a ligation reaction composition, wherein the at least one first adaptor comprises a first oligonucleotide comprising at least two ribonucleosides on the 3′

-end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5′

phosphate group and a fourth oligonucleotide that comprises a single-stranded portion when the third oligonucleotide and the fourth oligonucleotide are hybridized together,

ligating the at least one first adaptor and the at least one second adaptor to the RNA molecule to form a ligated product, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the same ligation reaction composition,

combining the ligated product with an RNA-directed DNA polymerase,

reverse transcribing the ligated product to form a reverse transcribed product,

digesting at least some of the ribonucleosides from the reverse transcribed product with a ribonuclease H (RNase H) to form an amplification template,

combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase to form an amplification reaction composition,

cycling the amplification reaction composition to form an amplified product, wherein the amplification reaction composition further comprises a reporter probe, a nucleic acid dye, or a reporter probe and a nucleic acid dye, and

detecting the amplified product, thereby detecting the RNA molecule.

17. A method for generating an RNA library comprising,

combining a multiplicity of different RNA molecules with a multiplicity of first adaptor species, a multiplicity of second adaptor species, and a double-strand specific RNA ligase to form a ligation reaction composition, wherein the at least one first adaptor comprises a first oligonucleotide comprising at least two ribonucleosides on the 3′

-end and a second oligonucleotide that comprises a single-stranded portion when the first oligonucleotide and the second oligonucleotide are hybridized together, and wherein the at least one second adaptor comprises a third oligonucleotide that comprises a 5′

phosphate group and a fourth oligonucleotide that comprises a single-stranded portion when the third oligonucleotide and the fourth oligonucleotide are hybridized together,

ligating the at least one first adaptor and the at least one second adaptor to the RNA molecule to form a multiplicity of different ligated product species, wherein the first adaptor and the second adaptor are ligated to the RNA molecule in the same ligation reaction composition,

combining the multiplicity of ligated product species with an RNA-directed DNA polymerase,

reverse transcribing at least some of the multiplicity of ligated product species to form a multiplicity of reverse transcribed product species,

digesting at least some of the ribonucleosides from at least some of the multiplicity of reverse transcribed products with a ribonuclease H (RNase H) to form a multiplicity of amplification template species,

combining the multiplicity of amplification template species with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase to form an amplification reaction composition,

cycling the amplification reaction composition to form a library comprising a multiplicity of amplified product species, wherein at least some of the amplified product species comprise an identification sequence that is common to at least some of the other amplified product species in the library.

18. The method of claim 17, wherein at least some of the forward primers, at least some of the reverse primers, or at least some of the forward primers and at least some of the reverse primers comprise an identification sequence or the complement of an identification sequence.