Abstract
Solutions and methods are disclosed for the effective, simple isolation/extraction of DNA, RNA and proteins from a single biological material sample, such as cells, tissues and biological fluids. The preferred solutions include effective amounts of a chaotropic agent(s), buffer, reducing agent, and may or may not include an organic solvent. Genomic DNA and total RNA can be isolated utilizing the solutions and methods of the invention in as little as 20 minutes, and proteins in as little as 30 minutes.
Filing date: Jul 31, 1995
Issue date: Aug 31, 1999
Inventor: Piotr Chomczynski
Primary Examiner: David S. Romeo
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What is claimed is:
1. A solution for isolating substantially pure and undegraded RNA, DNA and proteins from biological material, said solution comprising:
- at least one chaotropic agent,
- a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5.
- an organic solvent present at a concentration in the range of about 13 to about 23% (v/v) of said solution, and
- at least one chelating agent.
2. The solution of claim 1 wherein said chelating agent is selected from the group consisting of ethylenediamine tetraacetic acid and citric acid.
3. The solution of claim 1 further comprising a detergent.
4. The solution of claim 3 wherein said detergent is selected from the group consisting of sarcosine and polyoxyethylenesorbitan.
5. A method of isolating substantially pure and undegraded RNA, DNA and proteins from biological material, comprising the steps of:
- a) homogenizing a biological material sample in the solution of claim 2 to form an homogenate;
- b) recovering substantially pure, undegraded RNA from the homogenate by sedimentation;
- c) thereafter precipitating DNA in the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated DNA by one of sedimentation or spooling; and
- d) thereafter precipitating proteins from the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated proteins by sedimentation.
6. The method of claim 5 wherein said additional amount of organic solvent added to precipitate DNA is added to achieve a concentration in the range of about 28 to about 38% (v/v).
7. The method of claim 6 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
8. The method of claim 5 wherein said additional amount of organic solvent added to precipitate proteins is added to achieve a concentration in the range of about 78 to about 82% (v/v).
9. The method of claim 8 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
10. A method of isolating substantially pure and undegraded RNA from biological material, comprising the steps of:
- a) homogenizing a biological material sample in a solution of claim 1 to form an homogenate; and
- b) recovering substantially pure RNA from the homogenate by sedimentation.
11. A method of isolating substantially pure and undegraded DNA from biological material, comprising the steps of:
- a) homogenizing a biological material sample in a solution of claim 1 to form an homogenate; and
- b) precipitating DNA in the homogenate by adding an additional amount of an organic solvent thereto for a concentration of organic solvent in the range of about 28% to about 38% (v/v), and recovering the precipitated DNA by one of spooling and sedimentation.
12. The method of claim 11 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
13. A method of isolating substantially pure and undegraded RNA, DNA and proteins from biological material, comprising the steps of:
- a) homogenizing a biological material sample in a solution comprising:
- at least one chaotropic agent, and
- a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5, to form an homogenate;
- b) removing unhomogenized material from the homogenate by sedimentation;
- c) precipitating RNA in the remaining homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 13 to about 23% (v/v), and recovering the precipitated RNA by sedimentation;
- d) thereafter precipitating DNA in the remaining homogenate by adding an additional amount of organic solvent thereto, and recovering the precipitated DNA by one of spooling or sedimentation; and
- e) thereafter precipitating proteins from the remaining homogenate by adding an additional amount of an organic solvent thereto, and recovering the precipitated proteins by sedimentation.
14. The method of claim 13 wherein said organic solvent used for precipitating RNA is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethysulfoxide.
15. The method of claim 13 wherein said additional amount of organic solvent added to precipitate DNA is added to achieve a concentration in the range of about 28 to about 38% (v/v).
16. The method of claim 15 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
17. The method of claim 13 wherein said additional amount of organic solvent added to precipitate proteins is added to achieve a concentration in the range of about 78 to about 82% (v/v).
18. The method of claim 17 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
19. A method of isolating substantially pure and undegraded RNA from biological material, consisting essentially of the following steps:
- a) homogenizing a biological material sample in a solution consisting essentially of:
- at least one chaotropic agent, and
- a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 6 to about 7.5, to form an homogenate;
- b) removing unhomogenized material from the homogenate by sedimentation; and
- c) precipitating RNA in the homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 13 to about 23% (v/v), and recovering the precipitated RNA by sedimentation.
20. The method of claim 19 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.
21. A method of isolating substantially pure and undegraded DNA from biological material, consisting essentially of the following steps:
- a) homogenizing a biological material in a solution comprising:
- at least one chaotropic agent, and
- a buffer present in an amount sufficient to maintain the pH of said solution in the range of about 8 to about 12, to form an homogenate;
- b) removing unhomogenized material from the homogenate by sedimentation, and
- c) precipitating DNA in the homogenate by adding thereto an organic solvent to achieve a concentration in the range of about 28 to about 38% (v/v), and recovering the precipitated DNA by one of spooling or sedimentation.
22. The method of claim 21 wherein said organic solvent is selected from the group consisting of lower alcohols, acetone, polyethylene glycol and dimethylsulfoxide.