Small RNA capture, detection and quantification

• US 9,920,360 B2
• Filed: 11/04/2013
• Issued: 03/20/2018
• Est. Priority Date: 11/02/2012
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Wong, Linda, Chen, Caifu, Wu, Yalei, Dong, Shoulian, Liu, Chunmei
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US14/233,711
• Publication Number:
  US 20150105275A1
• Time in Patent Office: 1,597 Days
• Field of Search: 435 612, 435 911, 435 912
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

Methods, compositions and kits for capturing, detecting and quantifying mature small RNAs are provided herein. Embodiments of the methods comprise ligating 5′ and 3′ ligation adaptors to the 5′ and 3′ ends of the mature small RNAs, respectively, in the presence of 5′ and 3′ semi-degenerate ligation splints to generate a ligation product. Other embodiments comprise reverse transcribing polyadenylated mature small RNA with a universal reverse transcription primer and ligating an adaptor to the 3′ end of the cDNA in the presence of a semi-degenerate ligation splint to generate a cDNA ligation product.

Claims

1. A method for detecting a mature small RNA, the method comprising:

providing a sample comprising a mature small RNA;

polyadenylating the 3′

end of the mature small RNA;

reverse transcribing the polyadenylated mature small RNA using a universal reverse transcription primer, thereby forming a cDNA of the mature small RNA, wherein the universal reverse transcription primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal reverse primer portion;

ligating a universal ligation adaptor to the 3′

end of the cDNA, whereby a cDNA ligation product is formed, wherein the universal ligation adaptor comprises a ligation splint portion and further comprises a stem-loop structure;

amplifying the cDNA ligation product using a universal forward and reverse primer pair to form an amplification product; and

detecting the amplification product with a probe specific for the mature small RNA, wherein the ligating and the amplifying occur during overlapping intervals in the same reaction vessel.

2. The method of claim 1, wherein the polyadenylating step and the reverse transcribing step occur during overlapping intervals in the same reaction vessel.

3. The method of claim 1, wherein the universal ligation adaptor is selected from the group consisting of a DNA oligonucleotide and a DNA oligonucleotide comprising at least one blocking agent.

4. The method of claim 3, wherein the blocking agent is selected from the group consisting of 2′

-0-methyl, acridine, a minor groove binder (MGB), and an intercalating dye compound.

5. The method of claim 1, wherein the ligation splint portion comprises a 3′

single-stranded overhang which hybridizes with the 3′

region of the cDNA.

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

single-stranded overhang comprises about 3 to about 6 nucleotide bases.

7. The method of claim 1, wherein the ligation splint portion is a semi-degenerate.

8. The method of claim 7, wherein the semi-degenerate ligation splint portion comprises a 3′

terminal region comprising about 3 to about 6 degenerate nucleotide bases that hybridizes with the 3′

end of the cDNA and a 5′

region that hybridizes with the 5′

end of the universal ligation adaptor.

9. The method of claim 1, further comprising adding a blocking oligonucleotide to the ligating and/or amplifying step.

10. The method of claim 9, wherein the blocking oligonucleotide is selected from the group consisting of a 3′

-MGB blocking oligonucleotide, a 5′

-MGB blocking oligonucleotide, a 2′

-O-methyl blocking oligonucleotide, a 3′

-acridine blocking oligonucleotide, a 5′

-acridine blocking oligonucleotide, a STAR blocking oligonucleotide, and a blocking oligonucleotide comprising a poly(A) sequence.

11. The method of claim 1, wherein the universal ligation adaptor comprises a second portion complementary to the ligation splint portion.

12. The method of claim 11, wherein the complementarity between the second portion and the ligation splint portion of the universal ligation adaptor is located within the stem of the stem-loop structure.

13. The method of claim 1, wherein the universal ligation adaptor comprises at least one blocking agent at the 3′

end.

14. The method of claim 13, wherein the blocking agent is selected from the group consisting of 2′

-0-methyl, acridine, a minor groove binder (MGB), and an intercalating dye compound.

15. The method of claim 1, wherein the universal ligation adaptor is a DNA-RNA hybrid oligonucleotide.

16. The method of claim 1, wherein the universal ligation adaptor comprises a forward primer binding site.

17. The method of claim 1, wherein the forward primer binding site is located within the stem of the stem-loop structure.

18. The method of claim 1, wherein the forward primer binding site is located within the loop of the stem-loop structure.

19. The method of claim 1, wherein the forward primer binding site is located within some portion of both the stem and loop of the stem-loop structure.