Method for isolating extracellular nucleic acids using anion exchange particles

Patent metadata

Field	Value
Publication number	US-12031125-B2
Application number	US-202117385460-A
Country	US
Kind code	B2
Filing date	Jul 26, 2021
Priority date	Jun 10, 2015
Publication date	Jul 9, 2024
Grant date	Jul 9, 2024

Abstract

The present invention pertains to methods and kits for isolating extracellular nucleic acids from a biological sample using anion exchange particles. It was found that incorporating into the binding mixture a polyoxyalkylene fatty alcohol ether compensates performance variations that are attributable to differences in the anion exchange surface as they may occur e.g. between different lots/batches of the anion exchange particles and/or during storage of said particles. Moreover, including a polyoxyalkylene fatty alcohol ether in the binding mixture resulted in a higher purity of the obtained eluates revealing significantly less inhibition in a downstream reaction such as a PCR reaction.

First claim

The invention claimed is:

1. A method for isolating extracellular nucleic acids from a biological sample, comprising:
(a) preparing from the biological sample a binding mixture comprising
i) extracellular nucleic acids;
ii) particles providing an anion exchange surface;
iii) at least one non-ionic detergent selected from polyoxyalkylene fatty alcohol ethers; and
iv) optionally at least one salt and/or a buffer;

wherein the binding mixture has a pH so that extracellular nucleic acids bind to the particles, and
wherein the particles are not comprised in a column,
(b) separating the particles with the bound extracellular nucleic acids from the remaining binding mixture;
(c) optionally washing the bound extracellular nucleic acids; and
(d) optionally eluting bound extracellular nucleic acids.

2. The method according to claim 1 , wherein the binding mixture is prepared first by contacting the biological sample with a lysis and/or binding composition which comprises the at least one polyoxyalkylene fatty alcohol ether and optionally comprises a salt and/or a buffer.

3. The method according to claim 1 , wherein preparing the binding mixture further comprises contacting the biological sample with a proteolytic enzyme.

4. The method according to claim 1 , wherein the binding mixture is prepared by
forming a suspension by contacting the particles with a lysis and/or binding composition which comprises the at least one polyoxyalkylene fatty alcohol ether and optionally comprises a salt and/or a buffer;
contacting the suspension with the biological sample comprising extracellular nucleic acids; and
optionally adding a proteolytic enzyme prior to, at the same time as or after contacting the biological sample with the suspension.

5. The method according to claim 1 , wherein the binding mixture is incubated at conditions for the binding of extracellular nucleic acids, and the biological sample is lysed in the binding mixture.

6. The method according to claim 1 , wherein the binding mixture has a pH of ≤7.

7. The method according to claim 1 , wherein the binding mixture comprises a salt.

8. The method according to claim 7 , wherein
(a) the salt is an alkali metal salt or an ammonium salt;
(b) the salt is an alkali metal halide; or
(c) the salt is comprised in the binding mixture in a concentration of 50 mM to 1.5 M.

9. The method according to claim 1 , wherein
(a) the polyoxyalkylene fatty alcohol ether is a polyoxyethylene fatty alcohol ether;
(b) the chain length of the fatty alcohol component of the polyoxyalkylene fatty alcohol ether is 8 to 22 carbon atoms;
(c) the polyoxyalkylene fatty alcohol ether comprises a fatty alcohol component having from 14 to 22 carbon atoms and a polyoxyethylene component having from 2 to 150 (CH 2 CH 2 O) units;
(d) the polyoxyalkylene fatty alcohol ether is selected from the group consisting of polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and polyoxyethylene oleyl ether;
(e) the polyoxyalkylene fatty alcohol ether has a critical micelle concentration (CMC) of 0.15 mM or less; or
(f) the binding mixture comprises the polyoxyalkylene fatty alcohol ether in a concentration of 0.05% to 15% (w/v).

10. The method according to claim 1 , wherein the anion exchange particles are further not comprised in a device that would prevent the particles from moving in the binding mixture, and wherein the particles are collected from the binding mixture to recover the bound extracellular nucleic acids.

11. The method according to claim 1 , wherein
(a) the particles are magnetic;
(b) the particles have a mean diameter in a range of 100 nm to 10 μm; or
(c) the anion exchange surface of the particles comprises anion exchange moieties that provide anion exchange groups, wherein the anion exchange moieties are selected from the group consisting of monoamines, diamines, polyamines, nitrogen-containing aromatic or aliphatic heterocyclic groups, cyclic amines, aromatic amines and heterocyclic amines.

12. The method according to claim 1 , wherein the biological sample is:
(a) a cell-free, cell-depleted or cell-containing sample;
(b) selected from the group consisting of whole blood, plasma, serum, synovial fluid, pleural effusion, lymphatic fluid, urine, liquor, cerebrospinal fluid, ascites, milk, bronchial lavage, saliva, amniotic fluid, semen/seminal fluid, body fluids, body secretions, nasal secretions, vaginal secretions, wound secretions or excretions, and derived samples thereof;
(c) selected from the group consisting of whole blood, plasma and serum;
(d) a plasma sample;
(e) a stabilized sample;
(f) stabilized with a formaldehyde releaser; or
(g) a stabilized plasma sample.

13. The method according to claim 1 , wherein the method comprises
(a) preparing from the biological sample a binding mixture comprising
(i) extracellular nucleic acids;
(ii) magnetic particles providing an anion exchange surface;
(iii) at least one polyoxyethylene fatty alcohol ether in a concentration of 0.1% to 10% (w/v);
(iv) at least one alkali metal salt; and
(v) optionally at least one proteolytic enzyme;

wherein the binding mixture has a pH ≤6.5 so that extracellular nucleic acids bind to the magnetic particles,
(b) magnetically separating the magnetic particles with the bound extracellular nucleic acids from the remaining binding mixture;
(c) washing the bound extracellular nucleic acids; and
(d) eluting bound extracellular nucleic acids.

14. The method according to claim 1 , wherein the method comprises
(a) preparing from the biological sample a binding mixture comprising
(i) extracellular nucleic acids;
(ii) magnetic particles providing an anion exchange surface which comprises amine groups;
(iii) at least one polyoxyethylene fatty alcohol ether in a concentration of 0.1% to 6% (w/v), wherein the polyoxyethylene fatty alcohol ether is selected from the group consisting of polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and polyoxyethylene oleyl ether;
(iv) at least one alkali metal halide, in a concentration of 100 mM to 1 M; and
(v) optionally at least one proteolytic enzyme;

wherein the binding mixture has a pH ≤6.5 so that extracellular nucleic acids bind to the magnetic particles,
(b) magnetically separating the magnetic particles with the bound extracellular nucleic acids from the remaining binding mixture;
(c) washing the bound extracellular nucleic acids; and
(d) eluting bound extracellular nucleic acids.

15. The method according to claim 1 , wherein
(a) step (a) of claim 1 further comprises digesting the biological sample at room temperature to release the extracellular nucleic acids;
(b) steps (a) and (b) and optionally (c) and (d) of claim 1 are performed at room temperature; or
(c) a kit comprising the following components is used for performing the method:
(i) a lysis and/or binding composition comprising or preparing the binding mixture
(1) at least one polyoxyalkylene fatty alcohol ether;
(2) at least one salt; and
(3) at least one buffer;

wherein said composition has an acidic pH;
(ii) particles providing an anion exchange surface;
(iii) optionally a proteolytic enzyme as further component of the binding mixture;
(iv) optionally one or more wash solutions; and
(v) optionally one or more elution solutions.

16. A kit for performing the method according to claim 1 , comprising:
(a) a lysis and/or binding composition for preparing the binding mixture comprising
(i) at least one polyoxyalkylene fatty alcohol ether;
(ii) at least one salt; and
(iii) at least one b

Assignees

• Qiagen Gmbh

Inventors

• Wolf Alexander
• Hammerschmidt Sandra
• Voss Thorsten

Classifications

• C12Q1/6806

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

• C12N15/1013

  by using magnetic beads · CPC title

• C12N15/1006

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

• C12N15/101 Primary

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title