Methods of depleting a target nucleic acid in a sample and kits for practicing the same

Patent metadata

Field	Value
Publication number	US-9428794-B2
Application number	US-201314023354-A
Country	US
Kind code	B2
Filing date	Sep 10, 2013
Priority date	Sep 13, 2012
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

Provided are methods of depleting a target nucleic acid in a sample. The methods include contacting a target nucleic acid with two or more polymers that specifically hybridize to the target nucleic acid, and cleaving the hybridized regions of the target nucleic acid to deplete the target nucleic acid in the sample. Kits for practicing the subject methods are also provided.

First claim

What is claimed is:

1. A method of depleting an undesired target ribosomal ribonucleic acid (rRNA) in a sample and enriching for a desired RNA, the method comprising:
obtaining a total RNA sample comprising a desired RNA and an undesired target rRNA;
contacting the sample with a polymer cocktail comprising at least two synthetic oligonucleotides selected from SEQ ID NOs:06-177 that specifically hybridize to distinct regions of an undesired 18S target rRNA or an undesired 28S target rRNA in the sample to generate hybridized regions of the undesired target rRNA that are separated by a gap;
cleaving the hybridized regions to deplete the undesired target rRNA in the sample; and
collecting the desired RNA or removing the cleaved undesired target rRNA to enrich the for the desired RNA.

2. The method according to claim 1 , wherein the total RNA sample is isolated from a biological sample.

3. The method according to claim 2 , wherein the biological sample is selected from the group consisting of: a tissue sample, a cell sample, and a bacterial sample.

4. The method according to claim 3 , wherein the biological sample is a fixed sample.

5. The method according to claim 1 , wherein cleaving the hybridized regions comprises contacting the hybridized regions with an enzyme.

6. The method according to claim 5 , wherein cleaving the hybridized regions comprises contacting the hybridized regions with a ribonuclease.

7. The method according to claim 6 , wherein the ribonuclease specifically cleaves RNA present in an RNA/DNA duplex.

8. The method according to claim 6 , wherein the ribonuclease is RNase H.

9. The method according to claim 1 , wherein, after cleaving the hybridized regions, the method comprises removing the at least two synthetic oligonucleotides.

10. The method according to claim 9 , wherein the at least two synthetic oligonucleotides are removed by contacting the at least two synthetic oligonucleotides with an enzyme.

11. The method according to claim 10 , wherein the at least two synthetic oligonucleotides comprise deoxyuridine triphosphate (dUTP).

12. The method according to claim 11 , wherein the method comprises contacting the at least two synthetic oligonucleotides with uracil-N-glycosylase (UDG).

13. The method according to claim 9 , wherein the at least two synthetic oligonucleotides are removed by a separation protocol.

14. The method according to claim 13 , wherein the separation protocol comprises binding the at least two synthetic oligonucleotides to a solid support.

15. The method according to claim 1 wherein the undesired target rRNA comprises 5S rRNA, 5.8S rRNA or a combination thereof, the method further comprises:
contacting the sample with a synthetic oligonucleotide that specifically hybridizes to distinct regions of an undesired target 5S rRNA or an undesired target 5.8S rRNA in the sample to generate hybridized regions of the undesired target 5S rRNA or the undesired target 5.8S rRNA; and
cleaving the hybridized regions to deplete the undesired target 5S rRNA or the undesired target 5.8S rRNA in the sample.

16. The method according to claim 1 , further comprising:
contacting the sample with a synthetic oligonucleotide that specifically hybridizes to distinct regions of an undesired target mitochondrial ribonucleic acid (mtRNA) in the sample to generate hybridized regions of the undesired target mtRNA; and
cleaving the hybridized regions to deplete the undesired target mtRNA in the sample.

17. The method according to claim 1 further comprising:
contacting the sample with a synthetic oligonucleotide that specifically hybridizes to distinct regions of an undesired target transfer RNA (tRNA) in the sample to generate hybridized regions of the undesired target tRNA; and
cleaving the hybridized regions of the undesired target tRNA to deplete the undesired target tRNA in the sample.

18. The method according to claim 1 , further comprising:
contacting the sample with a synthetic oligonucleotide that specifically hybridizes to distinct regions of an undesired target deoxyribonucleic acid (DNA) in the sample to generate hybridized regions of the undesired target DNA; and
cleaving the hybridized regions of the undesired target DNA to deplete the undesired target DNA in the sample.

19. The method according to claim 18 , wherein cleaving the hybridized regions of the undesired target DNA comprises contacting the hybridized regions of the undesired target DNA with a deoxyribonuclease.

20. The method according to claim 1 , wherein the at least two synthetic oligonucleotides of the polymer cocktail comprise sequences that are complementary to unique regions of eukaryotic 28S or 18S ribosomal RNA.

21. The method according to claim 1 , wherein the at least two synthetic oligonucleotides of the polymer cocktail are sufficiently small to be removed by a nucleic acid purification step.

22. The method according to claim 21 , wherein the at least two synthetic oligonucleotides of the polymer cocktail are 11 to 30 or 31 to 40 nucleotides in length.

Assignees

• Clontech Lab Inc
• Takara Bio Usa Inc

Inventors

• Farmer Andrew
• Oglesby Ian
• Bostick Magnolia
• Oh Steve

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2537/159

  Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions · CPC title