lSOLATION OF NUCLEIC ACID

• US 20080300396A1
• Filed: 03/24/2008
• Published: 12/04/2008
• Est. Priority Date: 12/12/1994
• Status: Active Grant

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Larsen, Frank, DEGGERDAL, ARNE Helge
• Granted Patent: US 7,989,614 B2
• Time in Patent Office: Days
• US Class Current: 536/25.41
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The present invention provides a method of isolating nucleic acid from a sample, said method comprising contacting said sample with a detergent and a solid support, whereby soluble nucleic acid in said sample is bound to the support, and separating said support with bound nucleic acid from the sample. Where the method of the invention is used to isolate DNA, it may conveniently be couple with a further step to isolate RNA from the same sample.

Claims

1. A method of isolating genomic DNA from a sample, said method comprising (a) contacting said sample with a detergent and magnetic particles, said magnetic particles comprising an organic polymer, and whereby soluble genomic DNA in said sample is bound to the surface of said particles in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent, and (b) separating said magnetic particles with bound genomic DNA from the sample.

2. The method as claimed in claim 1, further comprising disrupting or lysing structural components or cells in the sample prior to the contacting step.

3. The method as claimed in claim 1, wherein the detergent is anionic.

4. The method as claimed in claim 3, wherein the detergent is sodium dodecyl sulphate, or another alkali metal alkylsulphate salt, or sarkosyl.

5. The method as claimed in claim 1, wherein the concentration of detergent is 0.2 to 30% (w/v).

6. The method as claimed in claim 1, wherein the detergent is contained in a composition additionally comprising one or more monovalent cations, chelating agents or reducing agents.

7. The method as claimed in claim 1, wherein the detergent is used in alkaline solution.

10. The method as claimed in claim 1, wherein the magnetic particle has a negatively charged, neutral or hydrophobic surface.

11. The method as claimed in claim 1, wherein the genomic DNA is eluted from the support, following separation from the sample.

12. The method as claimed in claim 11, wherein the genomic DNA is eluted by heating.

14. The kit as claimed in claim 22 or 24 further comprising one or more buffers, salts, lysis agents, chelating agents and/or reducing agents.

15. The method as claimed in claim 1, wherein the organic polymer is polyurethane.

16. The method as claimed in claim 1, wherein the organic polymer is polystyrene.

17. The method as claimed in claim 1, wherein the organic polymer is latex.

18. The method as claimed in claim 1, wherein the magnetic particle comprises superparamagnetic polystyrene beads.

19. The method as claimed in claim 1, wherein the magnetic particle is porous.

20. The method as claimed in claim 1, the method further comprising the step of detecting, hybridizing, amplifying or quantifying the bound genomic DNA after the separating step.

21. The method of claim 2, wherein the disrupting step is effected by one or more of grinding, heating, or alkaline lysis, of the sample.

22. A kit for isolating genomic DNA from a sample, the kit comprising (a) magnetic particles as defined in claim 1;

(b) one or more detergents; and

(c) instructions for isolating genomic DNA according to the method of claim 1.

23. A method of isolating RNA and genomic DNA from a sample, said method comprising (a) isolating soluble genomic DNA from said sample using magnetic particles according to the method of claim 1;

(b) isolating RNA from said sample.

24. A kit for isolating RNA and genomic DNA from a sample, the kit comprising (a) magnetic particles as defined in claim 23;

(b) oligo dT beads;

(c) one or more detergents; and

(d) instructions for isolating RNA and genomic DNA according to the method of claim 23.

25. The kit for isolating RNA and genomic DNA from a sample according to claim 24, wherein the magnetic particle is a superparamagnetic polystyrene bead.

8-9. -9. (canceled)

13. (canceled)

26. A method of isolating genomic DNA from cells in a sample, said method comprising (a) obtaining cells from said sample by immunomagnetic separation;

(b) producing a lysate by contacting said cells with a detergent and magnetic particles in the absence of any chaotropic agent, the magnetic particles comprising an organic polymer whereby soluble genomic DNA in said lysate is bound to the surface of the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent; and

(c) separating said magnetic particles with bound genomic DNA from said lysate.

27. The method as claimed in claim 26, wherein said cells are in a cell:

bead complex.

28. A method of isolating RNA and genomic DNA from cells in a sample, said method comprising (a) obtaining cells from said sample by immunomagnetic separation;

(b) producing a lysate by contacting said cells with a detergent and magnetic particles in the absence of any chaotropic agent, the magnetic particles comprising an organic polymer, and whereby soluble genomic DNA in said lysate is bound to the surface of the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent;

(c) separating said magnetic particles with bound genomic DNA from said lysate; and

(d) isolating RNA from said lysate.

29. The method as claimed in claim 28, wherein said cells are in a cell:

bead complex.

30. A method of isolating genomic DNA from a sample, said method comprising

(a) contacting said sample with a detergent and magnetic particles comprising an organic polymer wherein the surface of the magnetic particle has a negative, neutral or hydrophobic surface, and whereby soluble genomic DNA in said sample is bound to the surface of said particles in a sequence-independent manner;

and

(b) separating said magnetic particles with the bound genomic DNA from the sample.