Method for purifying a target nucleic acid

Patent metadata

Field	Value
Publication number	US-9624253-B2
Application number	US-201113810406-A
Country	US
Kind code	B2
Filing date	Jul 15, 2011
Priority date	Jul 15, 2010
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

The present invention pertains to method for purifying at least a target nucleic acid from a sample, said method comprising at least the following steps: a) incubating the sample with at least one protein-degrading compound; b) binding the target nucleic acid to a solid phase; c) eluting the target nucleic acid from the solid phase; d) incubating the eluted target nucleic acid with at least one protein-degrading compound; e) binding the target nucleic acid again to a solid phase; f) optionally eluting the bound target nucleic acid from the solid phase. It was surprisingly found that performing a second protein digestion step after the target nucleic acid was bound and eluted from a solid phase before the nucleic acids are rebound to a solid phase is very efficient in reducing remaining protein contaminations in the isolated nucleic acid.

First claim

The invention claimed is:

1. A method for purifying at least one target nucleic acid from a sample, comprising:
(A) incubating the sample with at least one protein-degrading compound,
(B) binding the target nucleic acid to a first solid phase,
(C) eluting the target nucleic acid from the first solid phase,
(D) incubating the eluted target nucleic acid with at least one protein-degrading compound, and
(E) binding the target nucleic acid again to a second solid phase,
wherein the protein degrading compound of steps (A) and (D) is a proteolytic enzyme; and
wherein binding in step (B), step (E), or both steps (B) and (E) are performed in the presence of an alcohol at a concentration of at least 30% v/v and at least one chaotropic agent.

2. The method of claim 1 , wherein the incubation in step (A), step (D), or both steps (A) and (D) are performed under conditions comprising one or more of the following
(a) heating,
(b) agitation,
(c) the presence of salts,
(d) the presence of chaotropic agents,
(e) a pH value of between 6 to 9, and
(f) an incubation period of at least 3 minutes.

3. The method of claim 1 , wherein binding in step (B), step (E), or both steps (B) and (E) are performed under conditions having one or more of the following characteristics:
(a) binding is performing in the presence of detergents,
(b) binding is performed under conditions that also promote binding of proteins to the solid phase, and
(c) binding is performed under conditions that promote binding of small nucleic acids.

4. The method of claim 3 , wherein the target nucleic acid comprises RNA.

5. The method of claim 1 , wherein one or more washing steps are performed between steps (B) and (C), after step (E), or both between steps (B) and (C) and after step (E).

6. The method of claim 5 , wherein the solution used for washing comprises one or more selected from the group consisting of chaotropic agents, alcohols, detergents, and buffering components.

7. The method of claim 1 , wherein the sample comprises at least one non-target nucleic acid and at least one target nucleic acid.

8. The method of claim 7 , wherein the non-target nucleic acid is DNA, and the target nucleic acid is RNA.

9. The method of claim 7 , further comprising removing non-target nucleic acids after step (A) and prior to step (B).

10. The method of claim 9 , further comprising enzymatically degrading the remaining non-target nucleic acids prior to step (D).

11. The method of claim 1 , wherein the sample is mixed with a nucleic acid stabilization composition, and wherein said stabilization composition comprises
(1) a cationic compound of the general formula:
Y + R 1 R 2 R 3 R 4 X −
wherein Y represents nitrogen or phosphor,
R 1 , R 2 , R 3 , and R 4 independently, represent a branched or unbranched C 1 -C 20 −alkyl group, a C 6 -C 20 aryl group and/or a C 6 -C 26 aralkyl group;
X − represents an anion of an inorganic or organic, mono-or polybasic acid; and

(2) at least one proton donor.

12. The method of claim 11 , wherein Y represents nitrogen.

13. A method for purifying RNA from a sample that comprises RNA and DNA, comprising:
(A) (1) incubating the sample with at least one proteolytic enzyme in the presence of a chaotropic agent and by heating the sample to at least 40° C.,
(2) removing at least a portion of the DNA by binding DNA to a first solid phase and separating the DNA bound to said first solid phase from the remaining sample comprising the RNA,

(B) (1) binding the RNA to a second solid phase in the presence of at least one chaotropic agent and alcohol in a concentration ≧30% v/v,
(2) performing at least one washing step for washing the RNA bound to said second solid phase,

(C) eluting the RNA from said second solid phase,
(D) incubating the eluted RNA with at least proteolytic enzyme in the presence of a chaotropic agent and by heating the sample to at least 40° C.,
(E) (1) binding the RNA again to a third solid phase in the presence of at least one chaotropic agent and alcohol in a concentration ≧30% v/v, and
(2) performing at least one washing step for washing the RNA bound to the solid phase.

14. The method of claim 1 , wherein total RNA, including small RNAs, is isolated as target nucleic acid, and wherein said method results in 95% of the isolated nucleic acid having an A 260/280 between 1.8 and 2.2.

15. The method of claim 1 , wherein the sample is a biological sample selected from body fluids, blood, blood products, tissue and bone marrow.

16. The method of claim 1 , wherein the solid phase capable of binding nucleic acids is selected from the group consisting of solid phases comprising or consisting of silica, magnetic silica particles, diatomaceous earth, glass, alkylsilica, aluminum silicate, borosilicate, nitrocellulose, hydroxyapatite, metal oxides, polymeric supports, membranes, and magnetic particles.

17. The method of claim 1 , further comprising:
(F) eluting the bound target nucleic acid from the second solid phase.

18. The method of claim 13 , further comprising:
(F) eluting the bound RNA from the third solid phase.

19. The method of claim 1 , wherein the target nucleic acid comprises small RNAs.

20. The method of claim 1 , wherein the first solid phase, the second solid phase, or both the first and second solid phases are beads.

21. The method of claim 1 , wherein the sample comprises blood.

22. The method according to claim 1 , wherein binding in step (B), step (E), or both steps (B) and (E) are performed in the presence of the alcohol at a concentration selected from the group consisting of 30% v/v to 90% v/v, 40% v/v to 90% v/v, 30% v/v to 80% v/v, 40% v/v to 80% v/v, 40% v/v to 70%, and 40% v/v to 65%.

23. The method according to claim 1 , wherein binding in step (B), step (E), or both steps (B) and (E) are performed in the presence of the at least one chaotropic agent at a concentration selected from the group consisting of 0.05M up to the saturation limit, 0.1M to 4M, and 1M to 4M.

Assignees

• Voss Thorsten
• Wyrich Ralf
• Qiagen Gmbh

Inventors

• Voss Thorsten
• Wyrich Ralf

Classifications

• C12N15/1006

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C07H1/08 Primary

  from natural products · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title