Isolation of nucleic acid

• US 7,173,124 B2
• Filed: 09/23/2005
• Issued: 02/06/2007
• Est. Priority Date: 12/12/1994
• Status: Expired due to Fees

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Invitrogen Dynal AS (Invitrogen Dynal Holding AS)
• Inventors: Deggerdal, Arne Helge, Larsen, Frank
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US11/234,001
• Publication Number:
  US 20060058519A1
• Time in Patent Office: 501 Days
• Field of Search: 536/25.4, 536/25.41, 536/25.42, 536/23.6
• US Class Current: 536/25.4
• 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 a solid support in the absence of any chaotropic agent, the solid support comprising an organic polymer, whereby soluble genomic DNA in said sample is bound to the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent, and (b) separating said support with bound genomic DNA from the sample.

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

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

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

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

6. A 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. A method as claimed in claim 1, wherein the detergent is used in alkaline solution.

8. A method as claimed in claim 1, wherein the solid support is particulate.

9. A method as claimed in claim 8, wherein the solid support comprises magnetic beads.

10. A method as claimed in claim 1, wherein the solid support has a hydrophobic surface.

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

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

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

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

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

18. A method as claimed in claim 1, wherein the solid support comprises superparamagnetic polystyrene beads.

19. A method as claimed in claim 1, wherein the solid support is porous.

20. A 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) a solid support as defined in claim 1;

(b) one or more detergents; and

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

13. A kit for isolating genomic DNA from a sample, the kit comprising superparamagnetic polystyrene beads and one or more detergents.

14. A kit as claimed in claim 13, further comprising one or more buffers, salts, lysis agents, chelating agents and/or reducing agents.

23. A method of isolating RNA and genomic DNA from a sample, said method comprising (a) contacting said sample with a detergent and a solid support in the absence of any chaotropic agent, the solid support comprising an organic polymer, whereby soluble genomic DNA in said sample is bound to the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent;

(b) separating said support with bound genomic DNA from the sample; and

(c) isolating RNA from said sample.

25. A kit for isolating RNA and genomic DNA from a sample, the kit comprising (a) a solid support comprising an organic polymer;

(b) one or more detergents; and

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

24. A kit for isolating RNA and genomic DNA from a sample, the kit comprising (a) superparamagnetic polystyrene beads;

(b) oligo dT beads; and

(c) one or more detergents.

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 a solid support in the absence of any chaotropic agent, the solid support comprising an organic polymer, whereby soluble genomic DNA in said lysate is bound to the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent; and

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

27. A 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 a solid support in the absence of any chaotropic agent, the solid support comprising an organic polymer, whereby soluble genomic DNA in said lysate is bound to the support in a sequence-independent manner in the presence of the detergent and absence of any chaotropic agent;

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

(d) isolating RNA from said lysate.

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

bead complex.