# US 7,608,399 B2 · Device and method for extraction and analysis of nucleic acids from biological samples

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- Number: US 7,608,399 B2
- Document ID: US7608399B2
- Status: Granted
- Kind: B2
- Application number: 11/768,076
- Filed: 2007-06-25
- Published: 2008-02-14
- Granted: 2009-10-27
- Earliest claimed: 2006-06-26
- Assignees: Blood Cell Storage, Inc.
- Inventors: Michael W. Reed; Oliver Z. Nanassy; Paul V. Haydock; Nigel Rudra Sharma; Ronald L. Bardell; Perry Hargrave
- Web page: https://patentgrep.com/patent/US7608399B2

## CPC

- C12Q 1/6851: Quantitative amplification
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01L 7/525: {with physical movement of samples between temperature zones}
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0887: Laminated structure
- B01L 2400/0457: passive flow or gravitation
- B01L 2400/0487: fluid pressure, pneumatics
- C12Q 1/686: Polymerase chain reaction [PCR]
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Device and methods for extracting and analyzing nucleic acids from biological samples.

## Claims

### Claim 1

A method for determining the quantity of nucleic acids in a sample, comprising:
(a) introducing a sample containing cells in a liquid medium into a first chamber of a vessel, the vessel comprising the first chamber and a second chamber, wherein the first chamber is in liquid communication with the second chamber and wherein the second chamber has at least a portion of one flat glass surface effective for binding nucleic acids;
(b) lysing the cells to provide nucleic acids in the liquid medium;
(c) transferring at least a portion of the liquid medium from the first chamber into the second chamber;
(d) extracting the nucleic acids from the liquid medium by binding the nucleic acids to the flat glass surface of the second chamber effective for binding nucleic acids to provide isolated nucleic acids;
(e) contacting the isolated nucleic acids with a fluorescent compound having a fluorescence intensity dependent on the concentration of nucleic acids; and
(f) measuring the fluorescence of the fluorescent compound to determine the quantity of isolated nucleic acids.

### Claim 2 (depends on 1)

The method of claim 1, wherein lysing the cells comprises contacting the cells with a chaotropic salt solution.

### Claim 3 (depends on 1)

The method of claim 1 further comprising sonicating or mechanically disrupting the cells in the first chamber.

### Claim 4 (depends on 1)

The method of claim 1 further comprising purifying the isolated nucleic acids by contacting the isolated nucleic acids with a chaotropic salt solution.

### Claim 5 (depends on 1)

The method of claim 1, wherein the fluorescent compound is immobilized in the second chamber.

### Claim 6 (depends on 1)

The method of claim 1 further comprising releasing the isolated nucleic acids from the surface of the second chamber effective for binding nucleic acids by contacting the isolated nucleic acids with a buffer solution before measuring the fluorescence of the fluorescent compound.

### Claim 7 (depends on 6)

The method of claim 6, wherein the buffer solution comprises the fluorescent compound.

### Claim 8 (depends on 1)

The method of claim 1, wherein the fluorescent compound is a minor groove binder or an intercalating agent.

### Claim 9 (depends on 1)

The method of claim 1, wherein the fluorescent compound has an emission maximum at a wavelength of 500 nm or less.

### Claim 10 (depends on 1)

The method of claim 1, wherein the fluorescent compound is a bis-benzamide compound.

### Claim 11 (depends on 1)

The method of claim 1, wherein the fluorescent compound is ethidium bromide.

### Claim 12 (depends on 1)

The method of claim 1, wherein the fluorescent compound is 2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium.

### Claim 13

A method for amplifying and quantifying the amount of nucleic acids in a sample, comprising:
(a) introducing a sample containing cells in a liquid medium into a first chamber of a vessel, the vessel comprising the first chamber and a second chamber, wherein the first chamber is in liquid communication with the second chamber and wherein the second chamber has at least a portion of one flat glass surface effective for binding nucleic acids;
(b) lysing the cells to provide nucleic acids in the liquid medium;
(c) transferring at least a portion of the liquid medium from the first chamber into the second chamber;
(d) extracting the nucleic acids from the liquid medium by binding the nucleic acids to a flat glass surface of the second chamber effective for binding nucleic acids to provide isolated nucleic acids;
(e) releasing the isolated nucleic acids from the surface of the second chamber effective for binding nucleic acids by contacting the isolated nucleic acids with a buffer solution to provide release nucleic acids;
(f) contacting the released nucleic acids with a nucleic acid amplification reaction mixture and a fluorescent compound having a fluorescence intensity dependent on the concentration of nucleic acids;
(g) treating the released nucleic acids and the nucleic acid amplification reaction mixture under conditions for amplifying the release nucleic acids to provide amplified nucleic acids; and
(h) measuring the fluorescence of the fluorescent compound to determine the quantity of amplified nucleic acids.

### Claim 14 (depends on 13)

The method of claim 13, wherein steps (g) and (h) are repeated a pre-determined number of times to determine the amount of amplified nucleic acids.

### Claim 15 (depends on 13)

The method of claim 13 further comprising contacting the released nucleic acids with a fluorescent compound having a fluorescence intensity dependent on the concentration of nucleic acids and measuring the fluorescence of the fluorescent compound to determine the quantity of isolated nucleic acids prior to step (f).

### Claim 16 (depends on 15)

The method of claim 15, wherein the fluorescent compound is immobilized in the second chamber.

### Claim 17 (depends on 13)

The method of claim 13, wherein the nucleic acid amplification mixture includes the fluorescent compound.

### Claim 18 (depends on 13)

The method of claim 13, wherein the buffer solution comprises the fluorescent compound and the nucleic acid amplification reaction mixture.

### Claim 19 (depends on 13)

The method of claim 13 further comprising determining the presence of a gene or gene product in the sample by the presence of amplified nucleic acids.

### Claim 20 (depends on 13)

The method of claim 13 further comprising identifying a type of a gene or gene product in a sample by the presence of amplified nucleic acids.

### Claim 21 (depends on 13)

The method of claim 13, wherein the released nucleic acids are transported from a first temperature zone of the second chamber to a second temperature zone of the second chamber.

### Claim 22 (depends on 21)

The method of claim 21, wherein the first temperature zone is at about 65° C. and the second temperature zone is at about 95° C.

### Claim 23 (depends on 13)

The method of claim 13, wherein the nucleic acid amplification reaction mixture comprises a first fluorescent compound having a fluorescence intensity dependent on the concentration of isolated nucleic acids and a second fluorescent compound having a fluorescence intensity dependent on the concentration of amplified nucleic acids.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first fluorescent compound is a minor groove binder or an intercalating agent.

### Claim 25 (depends on 23)

The method of claim 23, wherein the second fluorescent compound is an oligonucleotide probe.

### Claim 26 (depends on 23)

The method of claim 23, wherein the nucleic acid amplification reaction mixture further comprises a third fluorescent compound having a fluorescence intensity dependent on the concentration of amplified nucleic acids.

### Claim 27 (depends on 26)

The method of claim 26, wherein the third fluorescent compound is an oligonucleotide probe.

### Claim 28 (depends on 26)

The method of claim 26 further comprising measuring the fluorescence of the third fluorescent compound to determine the presence of a gene or gene product.

### Claim 29 (depends on 23)

The method of claim 23 further comprising measuring the fluorescence of the second fluorescent compound to determine the presence of a gene or gene product.

### Claim 30 (depends on 13)

The method of claim 13, wherein the fluorescent compound is a minor groove binder or an intercalating agent.

### Claim 31 (depends on 13)

The method of claim 13, wherein the fluorescent compound has an emission maximum at a wavelength of 500 nm or less.

### Claim 32 (depends on 13)

The method of claim 13, wherein the fluorescent compound is a bis-benzamide compound.

### Claim 33 (depends on 13)

The method of claim 13, wherein the fluorescent compound is ethidium bromide.

### Claim 34 (depends on 13)

The method of claim 13, wherein the fluorescent compound is [2-[N-(3-dimethylaminopropyl)-N-propvlamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-guinolinium].

### Claim 35 (depends on 13)

The method of claim 13, wherein treating the released nucleic acids with the nucleic acid amplification reaction mixture under conditions for amplifying the nucleic acids to provide amplified nucleic acids comprises amplification using the polymerase chain reaction.

### Claim 36 (depends on 13)

The method of claim 13, wherein treating the released nucleic acids with the nucleic acid amplification reaction mixture under conditions for amplifying the nucleic acids to provide amplified nucleic acids comprises amplification using isothermal DNA or RNA amplification methods.

### Claim 37 (depends on 13)

The method of claim 13, wherein the sample is derived from a blood product.

### Claim 38

A method for determining the microbial content of a blood product, the method comprising:
(a) introducing a sample of a blood product containing cells in a liquid medium into a first chamber of a vessel, the vessel comprising the first chamber and a second chamber, wherein the first chamber is in liquid communication with the second chamber, and wherein the second chamber has at least a portion of one flat glass surface effective for binding nucleic acids;
(b) contacting the cells in the liquid medium with a lysis buffer to provide nucleic acids in the liquid medium;
(c) transporting at least a portion of the liquid medium into the second chamber;
(d) extracting the nucleic acids from the liquid medium by binding the nucleic acids to the flat glass surface of the second chamber effective for binding nucleic acids to provide isolated nucleic acids;
(e) releasing the isolated nucleic acids from the flat glass surface of the second chamber effective for binding nucleic acids by contacting the isolated nucleic acids with an elution buffer to provide released nucleic acids;
(f) contacting the released nucleic acids with a nucleic acid amplification reaction mixture and a fluorescent compound having a fluorescence intensity dependent on the concentration of nucleic acids;
(g) measuring the fluorescence of the fluorescent compound to determine the quantity of released nucleic acids;
(h) treating the released nucleic acids and the nucleic acid amplification reaction mixture under conditions for amplifying the released nucleic acids by polymerase chain reaction to provide amplified nucleic acids; and
(i) measuring the fluorescence of the fluorescent compound to determine the quantity of amplified nucleic acids.

### Claim 39 (depends on 38)

The method of claim 38, wherein steps (h) and (i) are repeated a pre-determined number of times to determine the amount of amplified nucleic acids.

### Claim 40 (depends on 38)

The method of claim 38 further comprising contacting the isolated nucleic acids in the second chamber with a wash buffer and removing the wash buffer from the second chamber.

### Claim 41 (depends on 40)

The method of claim 40, wherein the wash buffer comprises a chaotropic salt solution.

### Claim 42 (depends on 38)

The method of claim 38, wherein the fluorescent compound is immobilized in the second chamber.

### Claim 43 (depends on 38)

The method of claim 38, wherein the lysis buffer comprises a chaotropic salt solution.

### Claim 44 (depends on 38)

The method of claim 38, wherein the elution buffer comprises the fluorescent compound and the nucleic acid amplification reaction mixture.