# US 7,264,927 B2 · Isolation and purification of nucleic acids

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- Number: US 7,264,927 B2
- Document ID: US7264927B2
- Status: Granted
- Kind: B2
- Application number: 10/244,144
- Filed: 2002-09-12
- Published: 2003-05-15
- Granted: 2007-09-04
- Earliest claimed: 2001-11-06
- Assignees: Cortex Biochem, Inc.
- Inventors: Ruhangiz D. Nargessi; Matt Pourfarzaneh
- Web page: https://patentgrep.com/patent/US7264927B2

## CPC

- C12N 1/08: Reducing the nucleic acid content
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 15/1017: {by filtration, e.g. using filters, frits, membranes}
- Y10T 436/25375: Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- Y10T 436/255: including use of a solid sorbent, semipermeable membrane, or liquid extraction
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A method for the isolation and purification of nucleic acids such as DNA, RNA, and PNA from various sources using cellulose particles or cellulose paper. Adjusting the concentrations of the salt and polyalkylene glycol to the levels that result in binding of nucleic acids to the cellulose particles or cellulose paper. Separating the nucleic acids bound to the cellulose particles or paper and eluting the nucleic acids from the particles or paper.

## Claims

### Claim 1

A method to bind nucleic acids to cellulose, wherein said nucleic acids have not been purified or semi-purified, said method comprising:
a) combining cellulose, wherein said cellulose is not chemically coupled or physically adsorbed to another material, with a solution containing said nucleic acids, thereby producing a combination, and
b) adjusting the salt and polyalkylene glycol concentrations of the combination to final concentrations suitable for binding the nucleic acids to the cellulose using a binding buffer having a concentration of from 0.5 M to 1.5 M salt and from 5% to 15% polyalkylene glycol, whereby all or a portion of the nucleic acids in the solution binds to the cellulose, and wherein the nucleic acids bind to the cellulose while the nucleic acids are in a non-aggregated state.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acids are DNA and the polyalkylene glycol is polyethylene glycol.

### Claim 3 (depends on 2)

The method of claim 2, wherein the polyethylene glycol has a molecular weight of from about 1000 to about 8000, and wherein the salt is sodium chloride.

### Claim 4 (depends on 2)

The method of claim 2, wherein the polyethylene glycol has a molecular weight of from about 6000 to about 8000, and wherein the salt is sodium chloride.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nucleic acids are RNA and the polyalkylene glycol is polyethylene glycol.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cellulose is in the form of particles.

### Claim 7

A method of separating nucleic acids from non-nucleic acid materials in a nucleic acid solution, wherein said nucleic acids have not been purified, said method comprising:
a) combining cellulose particles with a solution containing said nucleic acids and non-nucleic acid materials to produce a first combination;
b) adjusting the salt and polyethylene glycol concentrations of the first combination to concentrations suitable for binding said nucleic acids in the solution to the cellulose particles using a binding buffer having a concentration of from 0.5 M to 1.5 M salt and from 5% to 15% polyethylene glycol, producing a second combination comprising cellulose-bound nucleic acids, and wherein the nucleic acids bind to the cellulose while the nucleic acids are in a non-aggregated state;
c) separating the cellulose-bound nucleic acids from the second combination;
d) contacting the cellulose-bound nucleic acids separated in c) with an elution buffer to release the bound nucleic acids from the cellulose particles and into the elution buffer; and
e) separating the cellulose particles from the elution buffer to provide nucleic acids that are substantially free of the non-nucleic acid materials, wherein the separation of the cellulose particles in step c) and e) is carried out using a centrifuge or a column.

### Claim 8 (depends on 7)

The method of claim 7, wherein the separation of the cellulose particles in step c) and e) is carried out using a centrifuge.

### Claim 9 (depends on 7)

The method of claim 7, wherein the separation of the cellulose particles in step c) and e) is carried out using a column.

### Claim 10 (depends on 7)

The method of claim 7, wherein the nucleic acids bound to cellulose particles are DNA and are washed with a wash buffer, wherein the wash buffer removes impurities bound to the cellulose particles while leaving the DNA bound to the cellulose particles.

### Claim 11 (depends on 10)

The method of claim 10, wherein the DNA bound to the cellulose particles is eluted with an elution buffer that releases the DNA bound to the cellulose particles.

### Claim 12 (depends on 11)

The method of claim 11, wherein the DNA released by the elution buffer is isolated.

### Claim 13 (depends on 7)

The method of claim 7, wherein the polyethylene glycol has a molecular weight of about 1000 to about 8000, and wherein the salt is sodium chloride.

### Claim 14 (depends on 7)

The method of claim 7, wherein the polyethylene glycol has a molecular weight of about 6000 to about 8000, and wherein the salt is sodium chloride.

### Claim 15 (depends on 7)

The method of claim 7, wherein the nucleic acids and non-nucleic acid materials are obtained from a cell lysate.

### Claim 16 (depends on 15)

The method of claim 15, wherein the lysate is prepared from cells of human, animal, plant, viral or bacterial origin.

### Claim 17

A method to bind nucleic acids to cellulose paper, wherein said nucleic acids have not been purified or semi-purified, said method comprising:
a) combining cellulose paper with a solution containing said nucleic acids, thereby producing a combination, and
b) adjusting the salt and polyalkylene glycol concentrations of the combination to concentrations suitable for binding the nucleic acids to the cellulose paper using a binding buffer having a concentration of from 0.5 M to 1.5 M salt and from 5% to 15% polyalkylene glycol, whereby all or a portion of the nucleic acids in the solution binds to the cellulose paper, and wherein the nucleic acids bind to the cellulose while the nucleic acids are in a non-aggregated state.

### Claim 18 (depends on 17)

The method of claim 17, wherein the cellulose paper is from the group consisting of various thickness and combinations thereof.

### Claim 19 (depends on 17)

The method of claim 17, wherein the nucleic acids are DNA and the polyakylene glycol is polyethylene glycol.

### Claim 20 (depends on 17)

The method of claim 17, wherein the nucleic acids are RNA and the polyakylene glycol is polyethylene glycol.

### Claim 21 (depends on 19)

The method of claim 19, wherein the polyethylene glycol has an average molecular weight of from about 1000 to about 8000, and wherein the salt is sodium chloride.

### Claim 22 (depends on 19)

The method of claim 19, wherein the polyethylene glycol has an average molecular weight of from about 6000 to about 8000, and wherein the salt is sodium chloride.

### Claim 23

A method of separating nucleic acids from non-nucleic acid materials, wherein said nucleic acids have not been purified or semi-purified, said method comprising:
a) combining cellulose paper with a solution containing said nucleic acids and non-nucleic acid materials to provide a first combination;
b) adjusting the salt and polyethylene glycol concentrations of the first combination to concentrations suitable for binding said nucleic acids to the cellulose paper using a binding buffer having a concentration of from 0.5 M to 1.5 M salt and from 5% to 15% polyethylene glycol, producing a second combination comprising cellulose paper-bound nucleic acids, and wherein the nucleic acids bind to the cellulose while the nucleic acids are in a non-aggregated state;
c) separating the cellulose paper-bound nucleic acids from the second combination;
d) contacting the cellulose paper-bound nucleic acids separated in c) with an elution buffer to release the bound nucleic acids from the cellulose paper and into the elution buffer; and
e) separating the cellulose paper from the elution buffer to provide nucleic acids that are substantially free of the non-nucleic acid materials.

### Claim 24 (depends on 23)

The method of claim 23, wherein the separation of the cellulose paper in steps c) and e) is carried out manually or by centrifugation.

### Claim 25 (depends on 23)

The method of claim 23, wherein the separation of the cellulose paper in steps c) and e) is carried out by placing it in a column.

### Claim 26 (depends on 23)

The method of claim 23, wherein the nucleic acids bound to cellulose paper are washed with a wash buffer, wherein the wash buffer removes impurities bound to the cellulose paper while leaving the nucleic acids bound to the cellulose paper.

### Claim 27 (depends on 26)

The method of claim 26, wherein the nucleic acids bound to the cellulose paper are DNA and are eluted with an elution buffer, wherein the elution buffer releases the DNA bound to the cellulose paper.

### Claim 28 (depends on 27)

The method of claim 27, wherein the DNA released by the elution buffer is isolated.

### Claim 29 (depends on 23)

The method of claim 23, wherein the polyethylene glycol has an average molecular weight of about 1000 to about 8000, and wherein the salt is sodium chloride.

### Claim 30 (depends on 23)

The method of claim 23, wherein the polyethylene glycol has an average molecular weight of about 6000 to about 8000, and wherein the salt is sodium chloride.

### Claim 31 (depends on 23)

The method of claim 23, wherein the nucleic acids and non-nucleic acid materials are obtained from a cell lysate.

### Claim 32 (depends on 31)

The method of claim 31, wherein the lysate is prepared from cells of human, animal, plant, viral or bacterial origin.

### Claim 33

A method for binding nucleic acids to cellulose, comprising combining cellulose, wherein said cellulose is not chemically coupled or physically adsorbed to another material, with nucleic acids that have not been purified or semi-purified in a mixture containing sodium chloride at a concentration of from about 0.7 M to 2.5 M and a polyethylene glycol at a concentration of from about 5% to 10%, said concentrations being sufficient to bind said nucleic acids to said cellulose while the nucleic acids are in a non-aggregated state.