Method for facilitating an automated isolation of a biopolymer using magnetic particles

• US 8,729,252 B2
• Filed: 08/16/2004
• Issued: 05/20/2014
• Est. Priority Date: 08/29/2003
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Himmelreich, Ralf, Werner, Sabine
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US10/567,351
• Publication Number:
  US 20080132694A1
• Time in Patent Office: 3,564 Days
• Field of Search: None
• US Class Current: 536/25.4
• CPC Class Codes: C12N 15/1013 by using magnetic beads

Abstract

A method for isolating polynucleotides, such as DNA, RNA and hybrids thereof from an aqueous solution containing polynucleotides by reversibly binding the polynucleotides to silica-coated magnetic particles in the presence of a salt and non-ionic hydratable additive is disclosed. The salt and non-ionic hydratable additive concentrations are adjusted to levels that result in adhesion of the nucleic acid to the particles without degradation or precipitation of the nucleic acid.

Claims

1. A method for isolating a nucleic acid from an aqueous solution utilizing magnetic particles comprising the steps of:

a) adding silica-coated magnetic particles to an aqueous solution comprising the nucleic acid in the presence of a salt and a hydratable additive, which is selected from the group consisting of ethylene glycol, tetraethylene glycol, polyalkylene glycol, cyclodextrin, carageenan, dextran, dextran sulfate, xanthan, cellulose, hydroxypropyl cellulose, amylose, 2-hydroxypropyl-β

-cyclodextrin, agar agar or a mixture thereof, wherein the concentration of the additive is in the range of from 2% (w/v) to 7% (w/v) and is selected to prevent substantial clustering of the magnetic particles during the performance of the method and wherein the concentration of the salt is sufficiently high to cause the nucleic acid to adhere to the particles but not so high as to substantially denature or degrade the nucleic acid or to cause the nucleic acid to precipitate,

b) incubating the aqueous solution of step a) to allow the particles of step a) and the nucleic acid of step a) to adhere, reversibly bind or absorb to the particles of step (a),

c) applying a magnetic field to said solution to separate the magnetic particles from said solution,

d) optionally washing the separated magnetic particles of step c); and

e) exposing the magnetic particles from step (c) or (d) to an elution buffer under conditions in which said nucleic acid is eluted from said magnetic particles.

2. A method according to claim 1, wherein the nucleic acid is DNA, RNA or hybrid molecules of RNA and DNA.

3. A method according to claim 1, wherein the silica-coated magnetic particles are siliceous-oxide coated magnetic particles.

4. A method according to claim 1, wherein the salt is a chaotropic salt.

5. A method according to claim 4, wherein the chaotropic salt is selected from the group consisting of guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, lithium chloride, sodium perchlorate, sodium trichloroacetate and a mixture thereof.

6. A method according to claim 5, wherein the chaotropic salt is selected from the group consisting of guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride and a mixture thereof.

7. A method according to claim 1, wherein the salt is a non-chaotropic salt.

8. A method according to claim 7, wherein the non-chaotropic salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, calcium chloride, magnesium chloride and a mixture thereof.

9. A method according to claim 1, wherein the concentration of the salt in the aqueous solution of step a) is in a range of from 0.1 M to 10 M.

10. A method according to claim 1, wherein the polyalkylene glycol is polyethylene glycol, polypropylene glycol or a mixture thereof.

11. A method according to claim 10, wherein the polyalkylene glycol is polyethylene glycol.

12. A method according to claim 11, wherein the polyethylene glycol has a molecular weight in a range of from about 4000 g/mol to about 12000 g/mol.

13. A method according to claim 12, wherein the polyethylene glycol has a molecular weight in a range of from about 6000 g/mol to about 10000 g/mol.

14. A method according to claim 13, wherein the polyethylene glycol has a molecular weight of about 8000 g/mol.

15. A method according to claim 1, wherein the concentration of the additive in the aqueous solution of step a) is in a range of from 2% (w/v) to 7% (w/v).

16. A method according to claim 15, wherein the concentration of the additive in the aqueous solution of step a) is in a range of from 3% (w/v) to 6% (w/v).

17. A method according to claim 16, wherein the concentration of the additive in the aqueous solution of step a) is in a range of from 3% (w/v) to 5% (w/v).

18. A method according to claim 17, wherein the concentration of the additive in the aqueous solution of step a) is in a range of from 3% (w/v) to 4.8% (w/v).

19. A method according to claim 1, wherein the incubation in step b) is performed at room temperature for at least 30 seconds.

20. A method according to claim 1, further comprising the step of washing said magnetic particles at least once following step c) and prior to step d), under conditions in which the nucleic acid remains in the complex formed with said magnetic particles in step b).

21. A method according to claim 1, wherein the method for isolating a nucleic acid from a solution utilizing magnetic particles is an automated process.

22. The method of claim 1 wherein the nucleic acid is DNA.

23. The method of claim 22 wherein the hydratable additive is polyethylene glycol.

24. A kit for isolating a nucleic acid from an aqueous solution comprising at least:

a) an aliquot of silica-coated magnetic particles suspended in an aqueous solution in a first container,

b) a hydratable additive, a stock solution of a hydratable additive or a ready-for application solution of a hydratable additive, wherein said hydratable additive is selected from the group consisting of ethylene glycol, tetraethylene glycol, polyalkylene glycol, cyclodextrin, carageenan, dextran, dextran sulfate, xanthan, cellulose, hydroxypropyl cellulose, amylose, 2-hydroxypropyl-β

-cyclodextrin, agar agar and mixtures thereof, and/or

c) a chaotropic or a non-chaotropic salt, a stock solution of a chaotropic or a non-chaotropic salt or a ready-for-application solution of a chaotropic or non-chaotropic salt.

25. The kit of claim 24, further comprising:

a) an additive selected from the group consisting of ethylene glycol, polyalkylene glycol, cyclodextrin, carrageenan, dextran, dextran sulfate, xanthan, cellulose, hydroxypropyl cellulose, amylose, 2-hydroxypropyl-B-cyclodextrin, agar agar, glycerol, polyvinyl alcohol and a mixture thereof, and/or

b) a chaotropic salt, a stock solution of said salt or a ready-for-application solution of said salt in a container.

26. The kit of claim 25 wherein the additive is a stock solution or a ready-for application solution.

27. The kit of claim 24, wherein the silica-coated magnetic particles are siliceous-oxide coated magnetic particles.

28. The kit of claim 24 wherein the nucleic acid is DNA.