Methods for the separation of biological molecules using sulfolane

• US 8,686,129 B2
• Filed: 03/20/2007
• Issued: 04/01/2014
• Est. Priority Date: 03/20/2007
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Braman, Jeffrey C., Basehore, Lee S., Novoradovskaya, Natalia
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US11/688,652
• Publication Number:
  US 20080234474A1
• Time in Patent Office: 2,569 Days
• Field of Search: None
• US Class Current: 536/25.4
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Abstract

The present invention provides a method for the isolation of biological molecules by the adsorption of the molecules onto a mineral substrate in the presence of an appropriate mixture of salts and sufolane. Preferably, the biological molecules are nucleic acids. Compositions and kits for performing the process according to the invention are also provided.

Claims

1. A method for the separation of a nucleic acid from other biological molecules present in a sample, said method comprising:

applying an aqueous sample comprising at least one nucleic acid and at least one other biological molecule to at least one mineral substrate in the presence of one or more salts and sulfolane, wherein the concentration of the sulfolane is 15% to 80% by volume, and wherein the mineral substrate adsorbs at least one nucleic acid to create a mineral substrate-nucleic acid complex and a composition not adsorbed to the mineral substrate; and

removing at least a portion of the composition not adsorbed to the mineral substrate, thereby separating the nucleic acid from at least one other biological molecule present in the sample.

2. The method of claim 1, wherein the sample comprises DNA, RNA, or both.

3. The method of claim 2, wherein the sample further comprises a protein, and the protein is purified as a result of not binding to the mineral substrate.

4. The method of claim 1, wherein the concentration of the sulfolane is about 35% to 50% by volume.

5. The method of claim 1, wherein the mineral substrate-nucleic acid complex comprises DNA and RNA, and wherein the method further comprises digesting the DNA of the complex with DNase.

6. A method for the separation of DNA from RNA in a sample, said method comprising:

applying an aqueous sample comprising DNA and RNA to a first mineral substrate in the presence of one or more salts and in the absence of sulfolane, wherein the first mineral substrate retains predominantly the DNA and wherein the action of applying the aqueous sample results in a filtrate that is essentially devoid of the DNA, and

applying the filtrate to a second mineral substrate in the presence of one or more salts and sulfolane, wherein the concentration of the sulfolane is about 15% to 50% by volume, and wherein the action of applying the filtrate to the second mineral substrate results in binding of predominantly RNA,

thereby separating DNA in the sample from RNA in the sample.

7. The method of claim 6, wherein the concentration of the sulfolane is about 35% to 50% by volume.

8. The method of claim 6, wherein the DNA comprises genomic DNA.

9. The method of claim 8, wherein the genomic DNA is predominantly retained on the first mineral substrate.

10. The method of claim 9, further comprising recovering the genomic DNA from the first mineral substrate by eluting the genomic DNA from the first mineral substrate.

11. The method of claim 6, wherein the first mineral substrate and the second mineral substrate are the same.

12. The method of claim 6, further comprising, after applying the filtrate to the second mineral substrate, treating the second mineral substrate with DNase.

13. The method of claim 6, wherein the one or more salts present in the aqueous sample applied to the first mineral substrate and in the filtrate applied to the second mineral substrate comprise a chaotropic salt or a combination of guanidinium thiocyanate and at least one other chaotropic salt.

14. A method for the separation of RNA from other biological molecules present in a sample, said method comprising:

applying a sample comprising RNA and at least one other biological molecule to at least one mineral substrate in the presence of one or more salts and sulfolane, wherein the sulfolane concentration is 15% to 80% by volume, and wherein the mineral substrate binds the RNA; and

removing at least a portion of the sample not adsorbed to the mineral substrate,

thereby separating the RNA from at least one other biological molecule present in the sample.

15. The method of claim 14, wherein the sulfolane concentration is about 35% to 50% by volume.

16. The method of claim 14, wherein the one or more salts comprises a chaotropic salt or a combination of guanidinium thiocyanate and at least one other chaotropic salt.

17. A composition comprising:

a mineral substrate; and

an aqueous composition comprising one or more salts and sulfolane, wherein the sulfolane concentration is 15% to 80% by volume.

18. The composition of claim 17, further comprising a nucleic acid.

19. The composition of claim 17, wherein the sulfolane concentration is about 35% to 50% by volume.

20. A kit comprising:

at least one mineral substrate; and

at least one container holding an aqueous composition comprising one or more salts and sulfolane, wherein the sulfolane concentration is 15% to 80% by volume.

21. The kit of claim 20, further comprising at least one container holding at least one nucleic acid.

22. The kit of claim 20, wherein the sulfolane concentration is about 35% to 50% by volume.