METHODS FOR THE SEPARATION OF BIOLOGICAL MOLECULES USING SULFOLANE

• US 20080234474A1
• Filed: 03/20/2007
• Published: 09/25/2008
• Est. Priority Date: 03/20/2007
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California
• Inventors: Basehore, Lee S., Braman, Jeffrey C., Novoradovskaya, Natalia
• Granted Patent: US 8,686,129 B2
• Time in Patent Office: Days
• US Class Current: 536/25.3
• 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 isolation of a biological molecule, said method comprising:

applying a sample comprising at least one biological molecule to at least one mineral substrate in the presence of an appropriate mixture of salts and sulfolane, whereby said mineral substrate adsorbs at least one biological molecule.

2. The method of claim 1, wherein said biological molecule is a nucleic acid.

3. The method of claim 1, wherein said sample comprises a protein, and the protein is purified as a result of not binding to the mineral substrate.

4. A method for the isolation of single-stranded nucleic acids, said method comprising:

applying a sample comprising at least one single-stranded nucleic acid molecule to at least one first mineral substrate in the presence of an appropriate mixture of salts whereby said mineral substrate retains predominantly double-stranded nucleic acids and said applying results in a filtrate that is essentially devoid of double-stranded nucleic acids, and

applying the filtrate to a second mineral substrate in the presence of an appropriate mixture of salts and sulfolane, whereby said second mineral substrate binds predominantly single-stranded nucleic acids;

washing said second mineral substrate at least once with a low salt buffer comprising an organic solvent or a mixture of organic solvents; and

eluting said single-stranded nucleic acids from said second mineral substrate.

5. The method of claim 4, comprising recovering genomic DNA from said first mineral substrate.

6. The method of claim 4, comprising washing the first mineral substrate at least once with a low salt buffer comprising an organic solvent or a mixture of organic solvents.

7. The method of claim 4, comprising digesting contaminating DNA on the second mineral substrate with DNase.

8. The method of claim 4, wherein the sulfolane concentration is 35% to 45% by volume.

9. The method of claim 4, wherein said appropriate mixture of salts comprises guanidinium thiocyanate or a combination of guanidinium thiocyanate and other chaotropic salts.

10. The method of claim 4, wherein the concentration of said appropriate mixture of salts is about 1 to about 8 Molar (M).

11. The method of claim 4, wherein said sample is cultured cells, tissue, whole blood, serum, plasma, urine, feces, bacteria, viruses, or mixtures comprising enzyme reactions.

12. A method for the isolation of double-stranded nucleic acids, said method comprising:

applying a sample comprising at least one double-stranded nucleic acid molecule to at least one mineral substrate in the presence of an appropriate mixture of salts and optionally sulfolane, whereby said mineral substrate binds at least one double-stranded nucleic acid molecule;

contacting said mineral substrate with RNase; and

eluting said double-stranded nucleic acid molecules from said mineral substrate.

13. The method of claim 12, wherein said appropriate mixture of salts comprises guanidinium thiocyanate or a combination of guanidinium thiocyanate and other chaotropic salts.

14. The method of claim 12, wherein the concentration of said appropriate mixture of salts is about 1 molar to about 8 M.

15. The method of claim 12, wherein said sample is cultured cells, tissue, whole blood, serum, plasma, urine, feces, bacteria, viruses, or mixtures comprising enzyme reactions.

16. The method of claim 12, comprising washing said mineral substrate at least once with a low salt buffer comprising an organic solvent or a mixture of organic solvents.

17. The method of claim 16, wherein the organic solvent concentration in said low salt buffer is 35% to 40% by volume.

18. A process for the separation of single-stranded nucleic acids from double-stranded nucleic acids by treatment of a biological source, said treatment comprising:

a) applying to a first mineral support an aqueous sample comprising material of said source under conditions whereby said first mineral support adsorbs or binds only one of said single- or double-stranded nucleic acids followed by, optionally, washing said first mineral support; and

b) applying to a second mineral support the other of said single- or double-stranded nucleic acids, which was not adsorbed or bound by said first mineral support, in an aqueous solution containing sulfolane.

19. The process of claim 18, wherein

the applying step to said first mineral support comprises adding to said aqueous sample salts and sulfolane in amounts such that the single-stranded, but not the double stranded, nucleic acids are adsorbed on or bound to the first mineral support, followed by, optionally, washing said first mineral support;

the double-stranded nucleic acids, which were not adsorbed on or bound to said first mineral support, are applied to said second mineral support in the presence of sulfolane in amounts such that the double-stranded nucleic acids are adsorbed on or bound to said second mineral support, followed by, optionally, washing said second mineral support; and

the single-stranded nucleic acids, double-stranded nucleic acids, or both are eluted from said first and second mineral supports, respectively.

20. The process of claim 19, wherein

the applying step to said first mineral support comprises adding said aqueous sample to materials that complex alkaline-earth metal ions, in the absence of sulfolane, such that double-stranded, but not single-stranded nucleic acids are adsorbed on or bound to said first mineral support,

the single-stranded nucleic acids, which were not adsorbed on or bound to said first mineral support, are applied to said second mineral support in the presence of sulfolane in amounts such that the single-stranded nucleic acids are adsorbed on or bound to said second mineral support, followed by optionally, washing said second mineral support, and

the double-stranded nucleic acids, single-stranded nucleic acids, or both are eluted from said first and second mineral supports, respectively.

21. The process of claim 18, wherein

the applying step to the first mineral support comprises adding to said aqueous sample wetting, washing, or dispersing agents, in the absence of sulfolane, such that said double-stranded nucleic acids are adsorbed on or bound to said first mineral support, followed by, washing said first mineral support,

the single-stranded nucleic acids, which were not adsorbed on or bound to said first mineral support, are applied to said second mineral support in the presence of sulfolane in amounts such that the single-stranded nucleic acids are adsorbed on or bound to said second mineral support, followed by optionally, washing said second mineral support, and

eluting the single-stranded, double-stranded nucleic acids, or both from said first and second mineral supports, respectively.

22. The process of claim 18, wherein

the applying step to said first mineral support comprises adding to said aqueous sample salts and sulfolane in amounts such that both the single-stranded and double-stranded nucleic acids are adsorbed on or bound to said first mineral support,

one of the single- or double-stranded nucleic acids is, selectively, first eluted from said first mineral support, followed by eluting the other of the single- or double-stranded nucleic acids, and

the one of the single- or double-stranded nucleic acids, which was first eluted from said first mineral support, is applied to said second mineral support under conditions whereby the nucleic acids first eluted from said first mineral support are adsorbed on or bound to said second mineral support, followed by eluting the nucleic acids from said second mineral support.

23. The process of claim 22, wherein said salts are present in concentrations of from 1 to 10 M.

24. The process of claim 18, further comprising, prior to applying the sample to said first mineral support, cells in said source containing the nucleic acids are lysed with chaotropic substances present in concentrations of from 0.1 to 10 M.

25. The process of claim 18 wherein said sulfolane is present in concentrations of from 1 to 90% by volume.

26. The process of claim 18, wherein said first and second mineral supports are independently porous or non-porous and comprised of metal oxides or mixed metal oxides, silica gel, glass particles, powdered glass, diatomaceous earth, quartz, alumina, zeolites, titanium dioxide, or zirconium dioxide, the particle size of said mineral supports is from 0.1 micrometers to 1000 micrometers, and the pore size of porous mineral supports is from 2 to 1000 micrometers.

27. A composition comprising a combination of one or more salts and sulfolane, which allows at least one biological molecule to adsorb to a mineral substrate.

28. The composition of claim 27, whereby said biological molecule is a nucleic acid.

29. The composition of claim 27, wherein the sulfolane concentration is 35 to 40% by volume.

30. A kit comprising at least one container holding a composition comprising a combination of at least one salt and sulfolane, which allows a biological molecule to adsorb to a mineral substrate.