BUFFER FOR ONE-STEP DNA EXTRACTION

• US 20130303746A1
• Filed: 05/08/2013
• Published: 11/14/2013
• Est. Priority Date: 05/09/2012
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Miller, Ingrid, Ruegg, Bryony
• Granted Patent: US 9,540,635 B2
• Time in Patent Office: Days
• US Class Current: 536/25.42
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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

DNA is extracted from epithelial cells and other cells without cell walls, by use of a DNA extraction buffer that contains a C₁-C₄ alcohol, a cell lysis detergent, and a buffering agent maintained at a slightly basic pH. The cells are immersed in the buffer and gently agitated at ambient temperature, and DNA extraction and precipitation occur in thirty minutes or less, and often in five minutes or less.

Claims

1. A process for extracting DNA from cells without cell walls, said process comprising:

(a) immersing said cells in an aqueous solution comprising a C₁-C₄ alcohol at from about 50% to about 85% by weight, a cell lysis detergent at from about 0.05% to about 0.5% by weight, and a buffering agent maintaining a pH of from about 7.5 to about 9.0, and substantially no protease, substantially no reducing agent, and substantially no non-detergent, inorganic salt, at a temperature of from about 15°

C. to about 35°

C. for from about five seconds to about thirty minutes, to cause DNA to be liberated from said cells and to precipitate; and

(b) recovering said DNA so precipitated from said aqueous solution.

2. The process of claim 1 wherein said C₁-C₄ alcohol is a member selected from the group consisting of methanol, ethanol, and isopropanol.

3. The process of claim 1 wherein said C₁-C₄ alcohol is isopropanol or a mixture of isopropanol and ethanol.

4. The process of claim 1 wherein said cell lysis detergent is a denaturing detergent.

5. The process of claim 1 wherein said cell lysis detergent is a member selected from the group consisting of anionic and zwitterionic detergents.

6. The process of claim 1 wherein said cell lysis detergent is an anionic detergent.

7. The process of claim 1 wherein said cell lysis detergent is a member selected from the group consisting of sodium dodecyl sulfate, sodium octadecyl sulfate, and sodium decyl sulfate.

8. The process of claim 1 wherein said cell lysis detergent is sodium dodecyl sulfate.

9. The process of claim 1 wherein said C₁-C₄ alcohol is a member selected from the group consisting of ethanol and isopropanol at a concentration of from about 60% to about 80% by weight, said cell lysis detergent is a member selected from the group consisting of sodium dodecyl sulfate, sodium octadecyl sulfate, and sodium decyl sulfate at a concentration of from about 0.1% to about 0.2% by weight, and said pH is from about 7.8 to about 8.5.

10. The process of claim 1 wherein said process consists of combining an aqueous suspension of said cells with a cell lysis buffer consisting of ethanol, isopropanol, or a mixture of ethanol and isopropanol, at a concentration of from about 75% to about 90% by weight, sodium dodecyl sulfate at a concentration of from about 0.1% to about 0.2% by weight, and said buffer is a member selected from the group consisting of tris(hydroxymethylamino)-methane at concentration of from about 6 mM to about 10 mM and a pH of from about 7.8 to about 8.5, balance water, in the total absence of any protease, reducing agent, or non-detergent, inorganic salt.

11. The process of claim 10 wherein said aqueous suspension and said cell lysis buffer are combined at a suspension:

buffer volumetric ratio of from about 1;

1 to about 1;

12.

12. The process of claim 10 wherein said aqueous suspension and said cell lysis buffer are combined at a suspension:

buffer volumetric ratio of from about 1;

2.5 to about 1;

6.

13. The process of claim 1 wherein step (a) is performed for a period of time ranging from about five seconds to about ten minutes.

14. The process of claim 1 wherein step (a) is performed for a period of time ranging from about five seconds to about five minutes.

15. A DNA extraction buffer comprising:

(i) a C₁-C₄ alcohol at a concentration of from about 75% to about 90% by weight,

(ii) a DNA extraction detergent at a concentration of from about 0.05% to about 0.5% by weight, and

(iii) a buffering agent maintaining a pH of from about 7.5 to about 9.0, in aqueous solution containing substantially no protease, substantially no reducing agent, and substantially no non-detergent, inorganic salt.

16. The DNA extraction buffer of claim 15 wherein said C₁-C₄ alcohol is a member selected from the group consisting of methanol, ethanol, and isopropanol.

17. The DNA extraction buffer of claim 15 wherein said C₁-C₄ alcohol is isopropanol.

18. The DNA extraction buffer of claim 15 wherein said DNA extraction detergent is a denaturing detergent.

19. The DNA extraction buffer of claim 15 wherein said DNA extraction detergent is a member selected from the group consisting of anionic and zwitterionic detergents.

20. The DNA extraction buffer of claim 15 wherein said DNA extraction detergent is an anionic detergent.

21. The DNA extraction buffer of claim 15 wherein said DNA extraction detergent is a member selected from the group consisting of sodium dodecyl sulfate, sodium octadecyl sulfate, and sodium decyl sulfate.

22. The DNA extraction buffer of claim 15 wherein said DNA extraction detergent is sodium dodecyl sulfate.

23. The DNA extraction buffer of claim 15 wherein said C₁-C₄ alcohol is a member selected from the group consisting of ethanol and isopropanol at a concentration of from about 60% to about 80% by weight, said DNA extraction detergent is a member selected from the group consisting of sodium dodecyl sulfate, sodium octadecyl sulfate, and sodium decyl sulfate at a concentration of from about 0.1% to about 0.2% by weight, and said pH is from about 7.8 to about 8.5, and said composition is devoid of proteases, reducing agents, and non-detergent, inorganic salts.