# US 5,346,999 A · Method of nucleic acid extraction

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- Number: US 5,346,999 A
- Document ID: US5346999A
- Status: Granted
- Kind: A
- Application number: 07/328,471
- Filed: 1989-03-24
- Granted: 1994-09-13
- Earliest claimed: 1985-01-18
- Assignees: Applied Biosystems, LLC
- Inventors: Guy R. Cathcart; Paul D. Grossman; P. Eric Mayrand; Eric S. Nordman; Norman M. Whiteley
- Web page: https://patentgrep.com/patent/US5346999A

## CPC

- C12N 9/0012: {acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)}
- 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)}
- 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)}
- C12M 47/12: {Purification (C12M47/04 takes precedence)}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- B01J 2219/00286: Reactor vessels with top and bottom openings
- B01J 2219/00308: interchangeably mounted in racks or blocks
- B01J 2219/00331: Details of the reactor vessels
- B01J 2219/00389: Feeding through valves
- B01J 2219/00488: by rotation of the reaction vessels
- B01J 2219/00689: using computers
- B01J 2219/00695: Synthesis control routines, e.g. using computer programs
- C40B 60/14: for creating libraries
- Y10T 436/25125: Digestion or removing interfering materials
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 5)

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

### Claim 8 (depends on 7)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 10)

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.

### Claim 12 (depends on 6)

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

### Claim 13 (depends on 6)

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

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 6)

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.

### Claim 16 (depends on 1)

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).

### Claim 17 (depends on 16)

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.

### Claim 18 (depends on 16)

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.

### Claim 19 (depends on 16)

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