Methods for detecting small RNA species

• US 20080241831A1
• Filed: 03/28/2007
• Published: 10/02/2008
• Est. Priority Date: 03/28/2007
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Chen, Jing
• Application Number:
  US11/729,755
• Publication Number:
  US 20080241831A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA

Abstract

The invention provides a method of detecting small target nucleotide sequences, in particular, small RNA species that are present in a sample. The method generally comprises a poly-A polymerization step or a ligation step to add a universal sequence to the 3′-end of all RNA molecules, followed by a universal primer-mediated cDNA synthesis, solid-phase selection, assay oligo annealing, extension and PCR amplification/labeling. The method of the invention can be practiced to amplify and label a small amount of miRNA or other ncRNA. The resulting amplification product can be read out on a universal array or an array with miRNA-specific or ncRNA-specific probes. The invention has multiple embodiments, including methods, compositions, and kits. In general, the nucleic acids, compositions, and kits comprise materials that are useful in carrying out the methods of the invention or are produced by the methods, and that can be used to detect small target nucleic acid sequences present in samples, in particular, small RNA species.

Claims

1. A method for determining the presence of a small target ribonucleotide sequence in a sample, said method comprising

(a) modifying ribonucleotide species contained in the sample by polyadenylating the 3′

ends;

(b) converting the polyadenylated ribonucleotide species into a plurality of complementary DNA (cDNA) sequences;

(c) immobilizing said plurality of cDNA sequences to a solid support;

(d) contacting said immobilized cDNA species with a pool of probe nucleic acids under conditions that allow sequence specific annealing, wherein each probe nucleic acid corresponds to a small target ribonucleotide sequence;

(e) extending said probe nucleic acids in a manner complementary to the immobilized cDNA species;

(f) removing the extended probe nucleic acids from the immobilized cDNA species;

(g) amplifying the extended probe nucleic acids to generate amplicons, and

(h) detecting said amplicons, wherein detection of each amplicon indicates the presence of a small target ribonucleotide sequence.

2. The method of claim 1, further comprising an initial step of enriching said sample for small RNA species.

3. The method of claim 2, wherein said small RNA species comprise ncRNA.

4. The method of claim 1, wherein said sample comprises purified RNA.

5. The method of claim 2, wherein said small target ribonucleotide sequences comprise micro RNA (miRNA).

6. The method of claim 1, wherein said miRNA sequences are less than 30 nucleotides.

7. The method of claim 1, wherein said polyadenylation step adds at least 15 nucleotides.

8. The method of claim 1, wherein the sample comprises purified small RNA species.

9. The method of claim 1, wherein said cDNA sequence is obtained using a labeled primer comprising a sequence complementary to the 3′

end of the polyadenylated ribonucleotide species and further comprising a label.

10. The method of claim 9, wherein said label has affinity for said solid support.

11. The method of claim 10, wherein said label comprises biotin.

12. The method of claim 1, wherein each probe nucleic acid comprises a unique address sequence and a universal primer sequence.

13. The method of claim 1, wherein said unique address sequence is complementary to a capture sequence on an array.

14. The method of claim 13, wherein said detection of the amplicons comprises capture on the array.

15. The method of claim 14, wherein said capture comprises binding of said unique address sequences to said capture sequences.

16. A method for determining the presence of a small target ribonucleotide sequence in a sample, said method comprising

(a) modifying ribonucleotide species in the sample by adding a chimera nucleic acid to the 3′

end of the ribonucleotide species, wherein said chimera nucleic acid comprises RNA bases at its 5′

end and DNA bases at its 3′

end;

(b) converting the modified ribonucleotide species into a plurality of complementary DNA (cDNA) sequences;

(c) immobilizing said plurality of cDNA sequences to a solid support;

(d) contacting said immobilized cDNA species with a pool of probe nucleic acids under conditions that allow sequence specific annealing, wherein each probe nucleic acid corresponds to a small target ribonucleotide sequence;

(e) extending said probe nucleic acids in a manner complementary to the immobilized cDNA species;

(f) removing the extended probe nucleic acids from the immobilized cDNA species;

(g) amplifying the extended probe nucleic acids to generate amplicons, and

(h) detecting said amplicons, wherein detection of each amplicon indicates the presence of a small target ribonucleotide sequence