# US 8,569,477 B2 · Method for isolating nucleic acids comprising the use of ethylene glycol multimers

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- Number: US 8,569,477 B2
- Document ID: US8569477B2
- Status: Granted
- Kind: B2
- Application number: 11/815,967
- Filed: 2006-02-13
- Published: 2009-03-12
- Granted: 2013-10-29
- Earliest claimed: 2005-02-11
- Assignees: Life Technologies Corporation
- Applicants: Erling Sigurd Finne
- Inventors: Erling Sigurd Finne
- Web page: https://patentgrep.com/patent/US8569477B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1013: {by using magnetic beads}
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a method of isolating nucleic acid from a sample, said method comprising contacting said sample with a solid support, preferably magnetic beads, in the presence of an ethylene glycol multimer consisting of from 2 to 70 ethylene oxide monomers, preferably tetraethylene glycol, whereby soluble nucleic acid in said sample is bound to the surface of the support, and separating said support with bound nucleic acid from the sample. Kits for performance of the invention are also provided.

## Claims

### Claim 1

A method of isolating DNA from a sample comprising:
providing i) a sample that comprises nucleic acids, ii) an ethylene glycol multimer consisting of 2 to 10 ethylene oxide monomers, iii) a salt comprising a monovalent or a divalent metal ion, and iv) a solid support having a hydrophilic surface;
contacting the sample with the solid support in the presence of the ethylene glycol multimer and the salt under conditions to allow binding of DNA in the sample to the surface of the solid support to form a solid support with bound DNA; and
separating the solid support with bound DNA from the sample, thereby isolating DNA from the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the multimer consists of 3 to 6 ethylene oxide monomers.

### Claim 3 (depends on 1)

The method of claim 1, wherein the multimer is an oligoethylene glycol with 10 or fewer ethylene oxide units.

### Claim 4 (depends on 1)

The method of claim 1, wherein the multimer is an oligoethylene glycol with between 2 and 6 ethylene oxide units.

### Claim 5 (depends on 1)

The method of claim 1, wherein the multimer is tetraethylene glycol.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method is performed in the presence of the ethylene glycol multimer at a final concentration of greater than 15%.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method is performed in the presence of the ethylene glycol multimer at a final concentration of 15-35%.

### Claim 8 (depends on 1)

The method of claim 1, wherein binding the DNA to the solid support is in the presence of the salt at a final concentration of less than 1M.

### Claim 9 (depends on 1)

The method of claim 1, wherein binding the DNA to the solid support is in the presence of the salt at a final concentration of 5 mM to 0.5M.

### Claim 10 (depends on 1)

The method of claim 1, further comprising binding the DNA to the solid support in the presence of alcohol at a final concentration of less than 30% (v/v).

### Claim 11 (depends on 1)

The method of claim 1, further comprising contacting the sample with a detergent, wherein the detergent is present at a final concentration of 0.2 to 30% (w/v).

### Claim 12 (depends on 11)

The method of claim 11, wherein the detergent is an anionic detergent.

### Claim 13 (depends on 11)

The method of claim 11, wherein the detergent is an alkali metal alkylsulfate salt.

### Claim 14 (depends on 1)

The method of claim 1, further comprising binding the DNA to the solid support in the presence of less than 1% (w/v) detergent and less than 30% (v/v) ethanol.

### Claim 15 (depends on 1)

The method of claim 1, wherein the contacting comprises contacting the sample and the solid support with a buffer solution, wherein the buffer solution consists essentially of a buffer and the ethylene glycol multimer.

### Claim 16 (depends on 1)

The method of claim 1, further comprising separating DNA molecules of greater than 100 base pairs from oligonucleotides of less than 30 nucleotides comprising contacting the sample with final concentration of less than 30% (v/v) ethanol.

### Claim 17 (depends on 1)

The method of claim 1, further comprising the step of eluting the DNA from the solid support with water or a low ionic strength medium.

### Claim 18 (depends on 17)

The method of claim 17, wherein the DNA is non-genomic DNA fragments of from 10 base pairs to 250 kb.

### Claim 19 (depends on 1)

The method of claim 1, wherein the sample is whole blood or a blood-derived product.

### Claim 20 (depends on 1)

The method of claim 1, wherein the sample is a sample produced by an amplification process.

### Claim 21 (depends on 1)

The method of claim 1, wherein the solid support is particulate.

### Claim 22 (depends on 1)

The method of claim 1, wherein the solid support comprises magnetic beads.

### Claim 23 (depends on 1)

The method of claim 1, wherein the solid support contains hydroxyl, epoxy, carboxylic acid, or sulfonic acid groups.

### Claim 24 (depends on 1)

The method of claim 1, further comprising washing the solid support after binding of the DNA to the solid support with a buffer solution or an ethanol solution.

### Claim 25 (depends on 24)

The method of claim 24, wherein washing the solid support after binding of the DNA comprises washing with a solution containing less than 250 mM salt and 50-90% ethanol, wherein the salt is selected from the group consisting of Mg2+, Ba2+, Ca2+, Na+, K+, and Li+salts.

### Claim 26 (depends on 1)

The method of claim 1, wherein the sample comprises cells, wherein the cells comprise DNA, and wherein the method further comprises lysing the cells to release DNA into the sample.

### Claim 27 (depends on 1)

The method of claim 1, wherein the method is performed at a pH of from about 4 to 9 and the solid support has a neutral or a negative charge at that pH range.

### Claim 28 (depends on 1)

The method of claim 1, wherein the salt is selected from the group consisting of Mg2+, Ba2+, Ca2+, Na+, K+, and Li+salts.

### Claim 29

A method for isolating nucleic acids from a sample comprising:
providing i) a sample that comprises DNA, ii) an oligoethylene glycol multimer comprising 2 to 6 ethylene oxide units, iii) a salt comprising a monovalent or a divalent metal ion at a concentration of less than 1M, and iv) a solid support having a hydrophilic surface;
contacting the sample with the solid support in the presence of the oligoethylene glycol multimer and the salt under conditions to allow binding of DNA in the sample to the surface of the solid support to form a solid support with bound DNA; and
separating the solid support with bound DNA from the sample comprising
removing the solid support with bound DNA from the sample, thereby isolating DNA from the sample.

### Claim 30 (depends on 29)

The method of claim 29 wherein the oligoethylene glycol multimer is tetraethylene glycol.

### Claim 31

A kit for isolating nucleic acids from a sample, the kit comprising:
i) a solid support having a hydrophilic surface;
ii) a salt comprising a monovalent or divalent metal ion; and
iii) an ethylene glycol multimer consisting of 2 to 10 ethylene oxide monomers,
wherein the nucleic acids comprise DNA.

### Claim 32 (depends on 31)

The kit of claim 31, wherein the solid support is particulate.

### Claim 33 (depends on 31)

The kit of claim 31, wherein the solid support comprises magnetic beads.

### Claim 34 (depends on 31)

The kit of claim 31, wherein the solid support contains hydroxyl, epoxy, carboxylic acid, or sulfonic acid groups.

### Claim 35 (depends on 31)

The kit of claim 31, wherein the ethylene glycol multimer is tetraethylene glycol.

### Claim 36 (depends on 31)

The kit of claim 31, wherein the salt is selected from the group consisting of Mg2+, Ba2+, Ca2+, Na+, K+, and Li+salts.