Method for isolating nucleic acids

Patent metadata

Field	Value
Publication number	US-9695465-B2
Application number	US-201214238625-A
Country	US
Kind code	B2
Filing date	Aug 13, 2012
Priority date	Aug 12, 2011
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

The present invention pertains to a method for isolating nucleic acids from a sample, preferably a blood sample, comprising the following steps: a) obtaining a sample which has been stabilized by the use of at least one cationic detergent, wherein the cationic detergent has formed complexes with the nucleic acids; b) obtaining the complexes optionally together with other sample components from the stabilized sample, wherein said complexes comprise the nucleic acids to be isolated; c) resuspending the complexes and optionally adding one or more additives before, during and/or after resuspension, thereby obtaining a resuspended sample comprising at least: i) the nucleic acid to be isolated; ii) at least one chaotropic agent; and iii) at least one chelating agent; and d) isolating nucleic acids from the resuspended sample. It was found that adding a chelating agent during resuspension considerably increases the nucleic acid yield as the formation of precipitates which irreversibly adhere to the container wall is considerably reduced.

First claim

The invention claimed is:

1. A method for isolating nucleic acids from a sample, comprising:
a) obtaining a sample that has been stabilised by the use of at least one cationic detergent, wherein the cationic detergent has formed complexes with the nucleic acids;
b) obtaining the complexes optionally together with other sample components from the stabilised sample, wherein said complexes comprise the nucleic acids to be isolated;
c) resuspending the complexes and optionally adding one or more additives before, during and/or after resuspension, thereby obtaining a resuspended sample comprising at least
i) the nucleic acid to be isolated;
ii) at least one chaotropic agent; and
iii) at least one chelating agent; and

d) isolating nucleic acids from the resuspended sample
wherein a plurality of samples is prepared according to steps a) to c) thereby providing a plurality of resuspended samples, wherein the resuspended, samples are divided into batches, the nucleic acids are isolated from the batches according to step d), and holding times between steps c) and d) vary at least between two batches.

2. The method according to claim 1 , wherein the sample is a blood sample.

3. The method according to claim 1 , wherein the resuspended sample is put on hold between step c) and step d) for at least 0.2 h, at least 0.3 h, at least 0.4 h, at least 0.5 h, at least 0.75 h or at least 1 h and/or for a time period of 0.5 h to 12 h, 1 h to 10 h, 1.5 h to 8 h, 2 h to 7 h or 3 h to 6 h.

4. The method according to claim 1 , wherein the resuspension performed in step c) has one or more of the following characteristics:
a) a resuspension solution is added wherein said resuspension solution comprises a non-chaotropic salt;
b) the chelating agent is added before, during, or after resuspension;
c) the chelating agent is added separately from the resuspension solution;
d) the chelating agent is comprised in a resuspension solution;
e) the chelating agent is added in a concentration so that the resuspended sample comprises the chelating agent in a concentration selected from 0.5 mM to 75 mM, 1 mM to 50 mM, 2.5 mM to 25 mM, and 5 to 15 mM;
f) the chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA); and/or
g) at least one additive is added before, during, or after resuspension that is selected from the group consisting of chaotropic agents, protein-degrading compounds and buffering agents, and thereby is comprised in the resuspended sample.

5. The method according to claim 4 , wherein the non-chaotropic salt of a) is an ammonium salt.

6. The method according to claim 1 , wherein the chelating agent present in the resuspended sample
a) reduces binding of the presipitates in the resuspended sample to the container comprising the sample;
b) increases the yield of the isolated nucleic acid; and/or
c) reduces variations in the nucleic acid isolation efficiency or quantity attributable to different holding times between step c) and d).

7. The method according to claim 1 , wherein said method has with respect to the chaotropic agent comprised in the resuspended sample one or more of the following characteristics:
a) the concentration of the chaotropic agent in the resuspended sample is selected from the group consisting of 0.1 M to 4 M, 0.5M to 3M and 0.75M to 2.5M;
b) the chaotropic agent is added in step c) in form of a separate solution;
c) the chaotropic agent is added in step c) after resuspension of the complexes and thereby is comprised in the resuspended sample; and/or
d) the chaotropic agent present in step c) is selected from the group consisting of chaotropic salts, guanidinium hydrochloride, guanidinium thiocyanate (GTC), guanidinium isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, and urea.

8. The method according to claim 7 , wherein the concentration of the chaotropic agent in the resuspended sample is at least 1M.

9. The method according to claim 7 , wherein the chaotropic agent of characteristic d) is GTC or GITC.

10. The method according to claim 1 , wherein a protein degrading compound is added in step c).

11. The method according to claim 10 , wherein the protein degrading compound is a proteolytic enzyme optionally selected from the group consisting of proteinases, proteases, subtilisins and subtilases.

12. The method according to claim 11 , wherein the protein degrading compound of b) is proteinase K.

13. The method according to claim 1 , wherein step c) comprises
aa) adding a resuspension solution that comprises a non-chaotropic salt and a chelating agent, but no chaotropic agent, thereby resuspending the complexes,
bb) adding a chaotropic agent after resuspension of the complexes so that the chaotropic agent is comprised in the resuspended sample, and
cc) optionally adding a proteolytic enzyme after resuspension of the complexes so that the proteolytic enzyme is included in the resuspended sample.

14. The method according to claim 13 , wherein in step bb), the chaotropic agent is added to the resuspended complexes in form of an aqueous solution.

15. The method according to claim 1 , wherein the isolation performed in step d) comprises the following steps:
i) digesting and/or denaturing the resuspended sample;
ii) binding the nucleic acids to a solid phase using appropriate binding conditions;
iii) optionally washing the nucleic acids; and
iv) optionally eluting the nucleic acids.

16. The method of claim 15 , wherein step i) is performed by heating and/or agitating the resuspended sample in the presence of a proteolytic enzyme.

17. The method according to claim 1 , wherein the sample is a blood sample, and in step a), the blood sample is stabilised by contacting the blood sample with a stabilizing composition comprising
i) 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; and
X − represents an anion of an inorganic or organic, mono- or polybasic acid; and
ii) at least one proton donor.

18. The method according to claim 17 , wherein Y represents nitrogen.

19. The method according to claim 1 , wherein the sample is a blood sample, and in step a), the blood sample is stabilised by contacting the blood sample with a stabilizing composition comprising
(i) an amino surfactant having the following formula (2):
R 1 R 2 R 3 N(O) x   (2)

wherein
R 1 and R 2 each independently is H, C 1 -C 6 alkyl residue, C 6 -C 12 aryl residue, or C 6 -C 12 aralkyl residue,
R 3 is C 1 -C 20 alkyl group, C 6 -C 26 aryl residue, or C 6 -C 26 aralkyl residue, and
X is an integer of 0 and 1, and
(ii) an acid or acid salt.

20. The method according to claim 1 , having one or more of the following characteristics:
a) the nucleic acid is RNA;
b) step d) is performed using an automated system;
c) wherein a plurality of samples is processed manually up to step c), thereby providing a plurality of resuspended samples; and/or
d) at least RNA is isolated from a sample comprising RNA and DNA, wherein isolation step d) comprises:
obtaining the resuspended sample comprising a proteolytic enzyme and continuing the digestion o

Assignees

• Voss Thorsten
• Qiagen Gmbh

Inventors

• Voss Thorsten

Classifications

• C12N15/1003

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

• C12Q1/6806 Primary

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

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2527/127

  the enzyme inhibitor or activator used · CPC title