Method for isolating nucleic acids and protein from a single sample

• US 8,110,351 B2
• Filed: 01/16/2003
• Issued: 02/07/2012
• Est. Priority Date: 01/16/2002
• Status: Expired due to Fees

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Invitrogen Dynal AS (Invitrogen Dynal Holding AS)
• Inventors: Bosnes, Marie
• Primary Examiner(s): Kapushoc, Stephen
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US10/501,162
• Publication Number:
  US 20050239068A1
• Time in Patent Office: 3,309 Days
• Field of Search: 435/6.1, 435/7.1, 435/287.1, 435/287.2, 536/22.1
• US Class Current: 435/6.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

The present invention comprises a method of isolating nucleic acid and protein from the same sample with solid supports, wherein nucleic acid and protein components contained in the sample become bound to distinct solid supports. The invention also allows for kits for isolating nucleic acid and protein from the same sample and for use of the method of isolating nucleic acid and protein for the analysis and/or comparison of mRNA and/or protein expression and/or their correlation to genomic information.

Claims

1. A method of isolating nucleic acid and protein from each other in a single sample, said method comprising:

a) providing a sample that comprises nucleic acid components and protein components;

b) contacting the sample with a plurality of magnetic particulate solid supports comprising;

(i) contacting the sample of step b with a first magnetic particulate solid support under conditions wherein nucleic acid components bind to the first magnetic particulate solid supports in a sequence independent manner and the protein components remain substantially intact; and

simultaneously

(ii) contacting the sample of step b with a second magnetic particulate solid support distinct from the first magnetic particulate solid support, under conditions wherein protein components contained in the sample bind to the second magnetic particulate solid support through a chromatographic interaction and the nucleic acid components remain substantially intact; and

c) separating the first magnetic particulate solid support to which are bound nucleic acid components and the second magnetic particulate solid supports to which are bound protein components from unbound components in the sample, thereby isolating nucleic acid components and protein components that are substantially intact;

wherein the sample is not contacted with a chaotropic agent.

2. The method of claim 1, wherein the method comprises providing a sample that contains DNA and RNA components, and further comprises binding both DNA and RNA components to the first magnetic particulate solid support.

3. The method of claim 1, wherein the method comprises providing a sample that contains RNA components, and further comprises contacting the sample with a third magnetic particulate solid support, wherein the first, second and third magnetic particulate solid supports are distinct, and wherein RNA components bind to the third magnetic particulate solid support.

4. The method of claim 3, further comprising contacting the sample with the first magnetic particulate solid support and the third magnetic particulate solid support in separate steps.

5. The method of claim 3, further comprising isolating RNA components from the sample using an RNA-specified capture-probe carried by or attached to, or capable of binding to said first magnetic particulate solid support.

6. The method of claim 5, wherein said capture probe is or comprises a dT oligonucleotide or dU oligonucleotide.

7. The method of claim 1, wherein the method comprises isolating nucleic acid and protein components from the same sample.

8. The method of claim 1, wherein the method comprises providing a sample containing mRNA.

9. The method of claim 1, wherein the method comprises providing a sample containing genomic DNA.

10. The method of claim 1, wherein the method comprises isolating total RNA and/or the total DNA from the sample.

11. The method of claim 1, wherein the method comprises isolating the total nucleic acid component from the sample.

12. The method of claim 1, wherein the method comprises isolating the total protein component from the sample.

13. The method of claim 1, further comprising providing a sample selected from a food or allied product, and a clinical, environmental or biological sample.

14. The method of claim 1, further comprising subjecting the sample to a preliminary treatment step to free the nucleic acid and/or protein components from structures or entities in which they may be contained.

15. The method of claim 1, further comprising providing a sample that comprises one or more cell populations, and subjecting the sample to a cell isolation procedure prior to contacting said sample with said plurality of first and second magnetic particulate solid supports.

16. The method of claim 15, further comprising separately isolating one or more particular cell populations from the sample.

17. The method of claim 1 or claim 15, further comprising subjecting the sample, or a cell population isolated therefrom, to a cell lysis step prior to contacting said sample with said first magnetic solid particulate support, wherein the cell lysis step may be performed in the absence of a chaotropic agent.

18. The method of claim 17, further comprising subjecting the cell surface proteins of cells within or isolated from said sample to an in vitro modification procedure prior to the cell lysis step.

19. The method claim 17, further comprising subjecting the sample to a cell lysis step, wherein cell lysis and nucleic acid binding to the first magnetic particulate solid support occur simultaneously or concomitantly.

20. The method of claim 17 or 19, further comprising lysing the sample in the presence of a plurality of magnetic solid particles capable of binding cells, wherein the plurality of magnetic solid particles and the first magnetic particulate solid support are of different size.

21. The method of claim 1, wherein the sample is not divided at any stage of the method.

22. The method of claim 1, further comprising conducting a cell isolation, lysis, or preliminary treatment step conducted prior to contacting the sample with the first magnetic particulate solid support, and dividing the sample after the cell isolation, lysis, and/or preliminary treatment step.

23. The method of claim 1, wherein said sample is contacted with said plurality of magnetic particulate solid supports sequentially or simultaneously.

24. The method of claim 23, wherein in a first step DNA is isolated from said sample, in a second step RNA is isolated from said sample and in a third step, protein is isolated from said sample, and wherein said steps may be performed in any order.

25. The method of claim 1, further comprising isolating DNA components on the first magnetic particulate solid support selected from supports carrying surface carboxyl or hydroxyl groups, silica or silica-based supports, and supports having a polyamine coated surface.

26. The method of claim 1, further comprising binding nucleic acid components from the sample to the plurality of first magnetic particulate solid support in the presence of a detergent.

27. The method of claim 1, wherein the first magnetic particulate solid support has a positive or negative surface charge.

28. The method of claim 1, further comprising contacting the sample with the first magnetic particulate solid support in the presence of a plurality of solid magnetic particles, wherein the plurality of first magnetic particulate solid supports and the plurality of solid magnetic particles are of different size.