# US 8,771,948 B2 · Nucleic acid preparation compositions and methods

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- Number: US 8,771,948 B2
- Document ID: US8771948B2
- Status: Granted
- Kind: B2
- Application number: 13/262,624
- Filed: 2010-04-01
- Published: 2012-07-12
- Granted: 2014-07-08
- Earliest claimed: 2009-04-03
- Assignees: Sequenom, Inc.
- Applicants: Michele E. Wisniewski; William H. Kwong; Firouz Mohsenian; Jian-Hua Ding
- Inventors: Michele E. Wisniewski; William H. Kwong; Firouz Mohsenian; Jian-Hua Ding
- Web page: https://patentgrep.com/patent/US8771948B2

## CPC

- 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}
- C12Q 1/68: involving nucleic acids
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Provided herein are methods and compositions to extract and enrich by, physical separation or amplification, relatively short nucleic acids from a nucleic acid composition containing a high background of longer nucleic acids (e.g., host or maternal nucleic acids; genomic nucleic acid and the like).

## Claims

### Claim 1

A method for enriching relatively short nucleic acid from cell-free nucleic acid, which comprises:
(a) contacting cell-free nucleic acid from a substantially cell-free biological composition with a solid phase under association conditions, wherein:
(i) the nucleic acid of the biological composition comprises relatively short nucleic acid and relatively long nucleic acid,
(ii) the relatively short nucleic acid is 300 base pairs or less,
(iii) the relatively long nucleic acid is larger than 300 base pairs, and
(iv) the association conditions do not include polyethylene glycol; whereby the relatively short nucleic acid and the relatively long nucleic acid are associated with the solid phase;
(b) introducing the solid phase after (a) to dissociation conditions that comprise a volume exclusion agent and a salt, wherein:
(i) the salt is not a chaotropic salt, and
(ii) the relatively short nucleic acid preferentially dissociates from the solid phase under the dissociation conditions as compared to the relatively long nucleic acid, thereby yielding dissociated nucleic acid; and
(c) separating the dissociated nucleic acid from the solid phase, whereby the relatively short nucleic acid is enriched in the dissociated nucleic acid relative to in the cell-free nucleic acid from the substantially cell-free biological composition.

### Claim 2 (depends on 1)

The method of claim 1, wherein the substantially cell-free biological composition is from a pregnant female.

### Claim 3 (depends on 2)

The method of claim 2, wherein the substantially cell-free biological composition is blood serum.

### Claim 4 (depends on 2)

The method of claim 2, wherein the substantially cell-free biological composition is blood plasma.

### Claim 5 (depends on 2)

The method of claim 2, wherein the substantially cell-free biological composition is urine.

### Claim 6 (depends on 1)

The method of claim 1, wherein the solid phase further comprises an agent that confers a paramagnetic property to the solid phase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the solid phase has a net charge.

### Claim 8 (depends on 1)

The method of claim 1, wherein the dissociated nucleic acid comprises deoxyribonucleic acid (DNA).

### Claim 9 (depends on 1)

The method of claim 1, wherein the association conditions comprise a C1-C6 alkyl alcohol.

### Claim 10 (depends on 9)

The method of claim 9, wherein the alcohol is ethanol.

### Claim 11 (depends on 1)

The method of claim 1, wherein the association conditions comprise a salt.

### Claim 12 (depends on 11)

The method of claim 11, wherein the salt is a chaotropic salt, an ionic salt or combination thereof.

### Claim 13 (depends on 1)

The method of claim 1, wherein the relatively short nucleic acid is about 200 base pairs or less.

### Claim 14 (depends on 13)

The method of claim 13, wherein the relatively short nucleic acid is about 50 to about 180 base pairs.

### Claim 15 (depends on 1)

The method of claim 1, which further comprises washing the solid phase after (a).

### Claim 16 (depends on 1)

The method of claim 1, wherein the salt is an ionic salt.

### Claim 17 (depends on 16)

The method of claim 16, wherein the ionic salt is sodium chloride.

### Claim 18 (depends on 1)

The method of claim 1, wherein the volume exclusion agent comprises a polyalkyl alcohol, dextran, Ficoll, polyvinyl pyrrolidone or combination thereof.

### Claim 19 (depends on 18)

The method of claim 18, wherein the polyalkyl alcohol is a polyethylene glycol (PEG).

### Claim 20 (depends on 19)

The method of claim 19, wherein the PEG is PEG-8000.

### Claim 21 (depends on 19)

The method of claim 19 wherein the dissociation conditions comprise about 10% PEG.

### Claim 22 (depends on 1)

The method of claim 1, wherein the relatively short nucleic acid preferentially dissociates from the solid phase under the dissociation conditions as compared to the relatively long nucleic acid at a ratio of about 1.05 to about 5 relatively short nucleic acid to relatively long nucleic acid.

### Claim 23 (depends on 1)

The method of claim 1, wherein the relatively short nucleic acid is enriched about 10% to about 45% in the dissociated nucleic acid relative to in the cell-free nucleic acid from the substantially cell-free biological composition.