# US 8,465,966 B2 · Post protein hydrolysis removal of a potent ribonuclease inhibitor and the enzymatic capture of DNA

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- Number: US 8,465,966 B2
- Document ID: US8465966B2
- Status: Granted
- Kind: B2
- Application number: 12/117,505
- Filed: 2008-05-08
- Published: 2012-06-28
- Granted: 2013-06-18
- Earliest claimed: 2002-08-15
- Assignees: University of South Florida
- Applicants: Matt Ewert
- Inventors: Matt Ewert
- Web page: https://patentgrep.com/patent/US8465966B2

## CPC

- C07K 17/10: the carrier being a carbohydrate
- C07C 51/42: Separation; Purification; Stabilisation; Use of additives
- C07C 65/40: containing singly bound oxygen-containing groups
- C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)}
- 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}
- C12N 15/101: {by chromatography, e.g. electrophoresis, ion-exchange, reverse phase}
- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- C12Q 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
- C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/34: Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)}
- G01N 33/86: involving blood coagulating time {or factors, or their receptors}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention concerns compositions and methods of extracting infectious pathogens from a volume of blood. In one embodiment, the method includes the steps of creating a fibrin aggregate confining the pathogens and introducing a fibrin lysis reagent to expose the pathogens for analysis. The present invention also concerns materials and methods for removing aurintricarboxylic acid (ATA) from a sample.

## Claims

### Claim 1

A method for nucleic acid extraction from a sample which contains aurintricarboxylic acid (ATA), comprising contacting the sample with a composition comprising urea and diethylenetriaminepentaacetate (DTPA).

### Claim 2 (depends on 1)

The method according to claim 1, wherein the concentration of urea in the sample is from about 6.0M to about 7.5M.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the composition further comprises sodium citrate.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the composition further comprises sodium hydroxide.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the composition further comprises methyl 6-O—(N-heptylcarbamoyl)-α-D-glucopyranoside.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the composition further comprises proteinase K.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the composition further comprises ethylenediaminetetraacetate.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the pH of the sample is brought to about 8.0.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the method further comprises heating the sample.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the sample further comprises a chaotrophic salt.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the method further comprises contacting the sample with a chaotrophic salt.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the composition is a dry powder.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the method further comprises the prior step of producing the composition of urea and DTPA by heating a solution of urea and DTPA to between 400 and 600° C. before the composition is combined with a sample.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the urea and DTPA solution is heated for about four hours.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the nucleic acid extraction comprises binding and/or washing steps, and the reagents used for the binding and/or washing steps are heated to about 55° C.-65° C. before or while contacting the sample with the reagents used for binding and/or washing steps.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the method further comprises contacting the sample with urease.

### Claim 17 (depends on 16)

The method according to claim 16, wherein 1,000 to 100,000 units of urease are added per ml of sample.

### Claim 18 (depends on 1)

The method according to claim 1, wherein the sample is blood.