# US 9,040,679 B2 · Methods and compositions for extraction and storage of nucleic acids

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- Number: US 9,040,679 B2
- Document ID: US9040679B2
- Status: Granted
- Kind: B2
- Application number: 13/968,497
- Filed: 2013-08-16
- Published: 2013-12-19
- Granted: 2015-05-26
- Earliest claimed: 2012-04-30
- Assignees: General Electric Company
- Applicants: General Electric Company
- Inventors: Erik Leeming Kvam; Bing Li; Brian Christopher Bales
- Web page: https://patentgrep.com/patent/US9040679B2

## CPC

- B01J 20/24: Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A solid matrix for the extraction, stabilization, and storage of nucleic acids is provided. At least one protein denaturant, and at least one acid or acid-titrated buffer reagent are impregnated in a dry state therein the matrix; and the matrix is configured to provide an acidic pH on hydration. The matrix is configured to extract nucleic acids from a sample and stabilize the extracted nucleic acids, particularly RNA, in a dry format under ambient conditions for a prolonged period of time. Methods for collecting and recovering the nucleic acids stored in the dry solid matrix are also described.

## Claims

### Claim 1

A solid matrix, comprising:
at least one protein denaturant; and
at least one acid or acid-titrated buffer reagent impregnated therein in a dry state;
wherein the matrix is a non-dissolvable dry solid material and wherein the matrix is configured to provide an acidic pH upon hydration, extract nucleic acids from a sample, and preserve the nucleic acids in a substantially dry state at ambient temperature; and the acid-titrated buffer reagent generates a pH in a range from 3 to 6, wherein the matrix comprises cellulose, cellulose acetate, glass fiber or combinations thereof.

### Claim 2 (depends on 1)

The matrix of claim 1 is a solid phase extraction matrix.

### Claim 3 (depends on 1)

The matrix of claim 1, wherein the extracted and preserved nucleic acids comprise ribonucleic acids (RNA), deoxy ribonucleic acids (DNA) or a combination thereof.

### Claim 4 (depends on 1)

The matrix of claim 1, wherein the extracted and preserved nucleic acids comprise RNA.

### Claim 5 (depends on 4)

The matrix of claim 4, wherein the extracted and preserved RNA has an RNA integrity number (RIN) of at least 4.

### Claim 6 (depends on 1)

The matrix of claim 1, wherein the acid comprises acetic acid, citric acid, tartaric acid, phosphoric acid, hydrochloric acid, Tris(2-carboxyethyl) phosphine-hydrochloric acid (TCEP-HCl), oxidized Tris(2-carboxyethyl) phosphine- hydrochloric acid (TCEP-O-HCl), sulfuric acid, nitric acid, vanillic acid, 3-(N-morpholino)propanesulfonic acid or combinations thereof.

### Claim 7 (depends on 1)

The matrix of claim 1 further comprising a UV protectant, a free-radical scavenger, a chelator or combinations thereof.

### Claim 8 (depends on 7)

The matrix of claim 7, wherein the UV protectant or free-radical scavenger is selected from the group consisting of hydroquinone monomethyl ether (MEHQ), hydroquinone (HQ), toluhydroquinone (THQ), and ascorbic acid.

### Claim 9 (depends on 1)

The matrix of claim 1 further comprising an RNase inhibitor.

### Claim 10 (depends on 9)

The matrix of claim 9, wherein the RNase inhibitor comprises a triphosphate salt, pyrophosphate salt or combinations thereof.

### Claim 11 (depends on 9)

The matrix of claim 9, wherein the RNase inhibitor comprises vanadyl ribonucleoside complex (VRC), sodium pyrophosphate, or a commercially available RNase inhibitor.

### Claim 12 (depends on 9)

The matrix of claim 9, wherein the RNase inhibitor comprises sodium triphosphate.

### Claim 13 (depends on 1)

The matrix of claim 1 further comprising at least one reducing agent.

### Claim 14 (depends on 13)

The matrix of claim 13, wherein the reducing agent is selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-ME), tris(2-carboxyethyl)phosphine (TCEP), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl)and a combination thereof.

### Claim 15 (depends on 1)

The matrix of claim 1, wherein the matrix comprises cellulose, cellulose acetate, nitrocellulose, glass fiber or any combination thereof.

### Claim 16 (depends on 1)

The matrix of claim 1, wherein the matrix is porous.

### Claim 17 (depends on 1)

The matrix of claim 1, wherein the protein denaturant is selected from a group consisting of guanidinium hydrochloride, guanidinium thiocyanate, sodium thiocyanate, potassium thiocyanate, arginine, sodium dodecyl sulfate (SDS), urea and combinations thereof.

### Claim 18 (depends on 1)

The matrix of claim 1, wherein the sample is a biological sample.

### Claim 19

An RNA extraction matrix comprising:
a protein denaturant comprising a chaotropic agent, a detergent or combinations thereof; and
an acid or acid-titrated buffer reagent impregnated therein in a dry state,
wherein the matrix is a porous non-dissolvable dry material configured to provide a pH in a range from 3 to 6 upon hydration for extracting RNA and stabilizing the extracted RNA with a RIN of at least 4, wherein the matrix comprises cellulose, cellulose acetate, glass fiber or combinations thereof.

### Claim 20 (depends on 19)

The matrix of claim 19 further comprising a UV protectant or free-radical scavenger selected from the group consisting of MEHQ, HQ, THQ, ascorbic acid and combinations thereof.

### Claim 21

An RNA extraction matrix comprising:
a protein denaturant comprising a chaotropic agent, a detergent or combinations thereof;
an acid or acid-titrated buffer reagent; and
an RNase inhibitor comprising a triphosphate salt or pyrophosphate salt impregnated therein in a dry state, wherein the matrix comprises a porous non-dissolvable dry material configured to provide a pH between 3 to 6 upon hydration and stabilize RNA with a RIN value of at least 4, wherein the matrix comprises cellulose, cellulose acetate, glass fiber or combinations thereof.

### Claim 22

A method for extracting and storing nucleic acids from a sample, comprising:
providing the sample on a dry non-dissolvable solid matrix comprising;
a protein denaturant; and
an acid or acid-titrated buffer reagent;
generating an acidic pH upon hydration for extraction of nucleic acids from the sample, where the acidic pH is in a range from 3 to 6;
drying the matrix comprising the extracted nucleic acids; and
storing the extracted nucleic acids on the matrix in a substantially dry state at ambient temperature, wherein the matrix comprises cellulose, cellulose acetate, glass fiber or combinations thereof.

### Claim 23 (depends on 22)

The method of claim 22 further comprising recovering the nucleic acids from the matrix.

### Claim 24 (depends on 23)

The method of claim 23, wherein the recovery of the nucleic acids from the matrix comprises rehydrating the matrix in an aqueous solution, a buffer or an organic solution.

### Claim 25 (depends on 23)

The method of claim 23, wherein the recovery of the nucleic acids from the matrix comprises electroelution.

### Claim 26 (depends on 22)

The method of claim 22, wherein the sample is a biological sample.

### Claim 27 (depends on 26)

The method of claim 26, wherein the biological sample comprises blood, serum, tissue, saliva, or cells.

### Claim 28 (depends on 26)

The method of claim 26, wherein the sample is a cell extract, a tissue culture cell preparation, an impure nucleic acidor a purified nucleic acid.

### Claim 29 (depends on 22)

The method of claim 22, wherein the extracted nucleic acids comprise RNA, DNA or a combination thereof.

### Claim 30 (depends on 22)

The method of claim 22, wherein the extracted nucleic acids comprise RNA.

### Claim 31 (depends on 30)

The method of claim 30, wherein the extracted RNA has a RIN of at least 4.

### Claim 32 (depends on 22)

The method of claim 22, wherein the matrix further comprises a UV protectant, a free-radical scavenger, a chelator or combinations thereof.

### Claim 33 (depends on 32)

The method of claim 32, wherein the UV protectant or free-radical scavenger is selected from the group consisting of MEHQ, HQ, THQ, and ascorbic acid.

### Claim 34 (depends on 22)

The method of claim 22, wherein the matrix further comprises an RNase inhibitor.

### Claim 35 (depends on 22)

The method of claim 22, wherein the matrix comprises a porous cellulose paper.

### Claim 36 (depends on 22)

The method of claim 22, wherein the protein denaturant is selected from the group consisting of guanidinium hydrochloride, guanidinium thiocyanate, sodium thiocyanate, potassium thiocyanate, arginine, SDS, urea and combinations thereof.

### Claim 37 (depends on 22)

The method of claim 22, wherein the matrix further comprises a reducing agent is selected from the group consisting of DTT, 2-ME, TCEP, TCEP-HCl and a combination thereof.

### Claim 38 (depends on 22)

The method of claim 22, wherein the acid comprises acetic acid, citric acid, tartaric acid, phosphoric acid, hydrochloric acid, TCEP-HCl , TCEP-O-HCl , sulfuric acid, nitric acid, vanillic acid, 3-(N-morpholino)propanesulfonic acid or combinations thereof