Method of nucleic acid extraction

• US 5,346,999 A
• Filed: 03/24/1989
• Issued: 09/13/1994
• Est. Priority Date: 01/18/1985
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Grossman, Paul D., Nordman, Eric S., Cathcart, Guy R., Whiteley, Norman M., Mayrand, P. Eric
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): BHAT, NINA NMN
• Application Number:
  US07/328,471
• Time in Patent Office: 1,999 Days
• Field of Search: 435/315, 435/285, 435/287, 435/290, 204/234, 210/22, 422/10, 536/27, 536/25.41, 536/26.73, 536/27.12, 436/177, 436/175, 935/19, 935/20, 935/21
• US Class Current: 536/25.41
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00331 Details of the reactor vessels
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00488 by rotation of the reaction vessels
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12M 47/12 Purification C12M47/04 takes precedence
• 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
• CPC Class Codes: C12N 9/0012 acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

An automated apparatus is provided which implements a new method of extracting and purifying nucleic acids from cells without the use of centrifugation. In the method, a lysate is created by treating the cells with proteinase K in the presence of a lysis buffer having a high concentration of a salt. The lysate is mixed with a phenol-based solvent system, thereby creating an emulsion. The emulsion is heated to promote phase separation. Similarly, the rate of phase separation is also enhanced by increasing the surface area of the emulsion. Once the phase separation is complete, the lower organic phase is removed and the upper aqueous phase is repeatedly extracted with the phenol-based solvent a preselected number of times, and is finally extracted using chloroform. The remaining aqueous phase is then dialyzed to further purify and concentrate the nucleic acid solution. Two preferred embodiments of apparatus are presented to accomplish this extraction.

Claims

1. A method of extracting nucleic acids from cells comprising the steps:

(a) creating a lysate by treating said cells with proteinase K in the presence of a lysis buffer;

(b) mixing said lysate with a phenol-based solvent system, thereby creating an emulsion;

(c) heating said emulsion to a temperature greater than 35 degrees C. in the absence of mixing action and without centrifugation to promote separation of said emulsion into an aqueous phase containing said nucleic acids and an organic phase containing phenol and denatured proteins;

(d) removing said organic phase to leave a remaining aqueous phase containing said nucleic acids.

2. The method of claim 1 wherein the temperature in Step (c) is greater than 45 degrees C.

3. The method of claim 1 wherein the temperature in Step (c) is about 55 degrees C.

4. The method of claim 1 wherein Steps (b), (c), and (d) are repeated, in order, N times, where N is a number greater than or equal to 1.

5. The method of claim 4 wherein after Steps (b), (c), and (d) have been repeated N times, the remaining aqueous phase is mixed with chloroform sufficiently to create an emulsion and Steps (c) and (d) are repeated to leave an aqueous phase containing said nucleic acids.

6. The method of claim 5 wherein the remaining aqueous phase after mixing with chloroform is dialyzed.

7. The method of claim 5 wherein said lysis buffer comprises a detergent and a high concentration of a salt.

8. The method of claim 7 wherein said lysis buffer further comprises a chaotropic agent.

9. The method of claim 8 wherein said lysis buffer comprises a chelating agent.

10. The method of claim 9 wherein said lysis buffer further comprises a buffer.

11. The method of claim 10 wherein said lysis buffer comprises urea, NaCl having a molar concentration between 0.5 and 2, a detergent Tris-HCl, and EDTA.

12. The method of claim 6 wherein the temperature of Step (c) is about 55 degrees C.

13. The method of claim 6 wherein said phenol-based solvent system comprises a mixture of phenol, chloroform, and an antisurfactant.

14. The method of claim 13 wherein the antisurfactant is isoamyl and the ratios of phenol to chloroform to isoamyl alcohol are about 50:

48;

2.

15. The method of claim 6 wherein the lysis buffer comprises 8M urea, 1M NaCl, 1% SDS, 50 mM Tris-HCl, and 10 mM EDTA, pH 8.0.

16. The method of claim 1 further comprising the step:

(e) increasing the surface area of the emulsion thereby further promoting phase separation, Step (e) occuring after Step (c) but before Step (d).

17. The method of claim 16 wherein Steps (b), (c), (e), and (d) are repeated N times in that order, where N is a number greater than or equal to 1.

18. The method of claim 16 wherein after Steps (b), (c), (e), and (d) have been repeated N times, the remaining aqueous phase is mixed with chloroform thereby creating an emulsion, and Steps (c), (e), and (d) are repeated in that order to leave an aqueous phase containing said nucleic acids.

19. The method of claim 16 wherein the remaining aqueous phase after mixing with chloroform is dialyzed.