# US 2013/0280787 A1 · Extraction of nucleic acids from wax-embedded samples

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- Number: US 2013/0280787 A1
- Document ID: US20130280787A1
- Status: Published application
- Kind: A1
- Application number: 13/703,205
- Filed: 2011-06-14
- Published: 2013-10-24
- Earliest claimed: 2010-06-14
- Assignees: Qiagen GmbH
- Inventors: Markus Mueller; Thorsten Singer; Esther Rosenberg; Joerg Hucklenbroich
- Web page: https://patentgrep.com/patent/US20130280787A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- 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 relates to a method for extracting nucleic acids from a wax-embedded sample, the use of particular solvents for removing wax from a wax-embedded sample in a method for extracting, isolating and/or purifying nucleic acids from a crosslinked wax-embedded sample as well as to a kit for extracting, isolating and/or purifying nucleic acids from a wax-embedded sample.

## Claims

### Claim at position 1

1-15. (canceled)

### Claim 16

A method for extracting nucleic acids from a wax-embedded sample, comprising:
(1) contacting the sample with a wax-solubilizing agent comprising at least one organic solvent immiscible with water,
(2) optionally incubating the mixture obtained in step (1),
(3) adding an aqueous lysis buffer to the mixture still comprising the wax-solubilizing agent, and
(4) incubating the mixture obtained in step (3) to obtain a bi- or multi-phasic mixture, comprising at least an organic phase, said organic phase consisting essentially of the dissolved wax and the wax-solubilizing agent, and an aqueous phase, said aqueous phase comprising the nucleic acids, wherein steps (1) and (3) may as well be carried out as one combined step.

### Claim 17 (depends on 16)

The method according to claim 16, further comprising:
(5) optionally separating the organic phase from the aqueous phase, and
(6) isolating and/or purifying the nucleic acids from the aqueous phase.

### Claim 18 (depends on 16)

The method according to claim 16, wherein the sample is a paraffin embedded sample, and the wax-solubilizing agent comprises at least one organic solvent

### Claim 19 (depends on 18)

The method according to claim 18, wherein the paraffin embedded sample is a formalin-fixed paraffin-embedded sample (FFPE-sample).

### Claim 20 (depends on 18)

The method according to claim 18, wherein the at least one organic solvent is selected from the group consisting of linear, branched and cyclic C10-C16 alkanes and mixtures thereof.

### Claim 21 (depends on 18)

The method according to claim 18, wherein the at least one organic solvent is selected from the group consisting of C13-C16 alkanes and mixtures thereof.

### Claim 22 (depends on 18)

The method according to claim 18, wherein the at least one organic solvent comprises tetradecane, pentadecane or hexadecane.

### Claim 23 (depends on 16)

The method according to claim 16, wherein a dye/a colorant may be comprised in the wax-solubilizing agent.

### Claim 24 (depends on 16)

The method according to claim 16, wherein the step of incubating the mixture of the wax-solubilizing agent and the wax-embedded sample according to step (2) is carried out at a temperature in the range of from 15° C. to 85° C.

### Claim 25 (depends on 24)

The method according to claim 24, wherein the step of incubating the mixture of the wax-solubilizing agent and the wax-embedded sample according to step (2) is carried out at a temperature in the range of from 20° C. to 55° C.

### Claim 26 (depends on 25)

The method according to claim 25, wherein the step of incubating the mixture of the wax-solubilizing agent and the wax-embedded sample according to step (2) is carried out at a temperature in the range of from room temperature to 35° C.

### Claim 27 (depends on 26)

The method according to claim 26, wherein the step of incubating the mixture of the wax-solubilizing agent and the wax-embedded sample according to step (2) is carried out at room temperature.

### Claim 28 (depends on 24)

The method according to claim 24, wherein the step of incubating the mixture of the wax-solubilizing agent and the wax-embedded sample according to step (2) is carried out at a temperature in the range of from 15° C. to 85° C. for 1 second to 3 hours, for 10 seconds to 1 hour, from 20 seconds to 30 minutes, or from 30 seconds to 15 minutes.

### Claim 29 (depends on 16)

The method according to claim 16, wherein the lysis buffer comprises a buffering agent and a detergent.

### Claim 30 (depends on 29)

The method according to claim 29, wherein the buffering agent is selected from the group consisting of Trips, Mops, Mes, Hepes, phosphates, borates and carbonates, and the detergent is selected from the group consisting of anionic and zwitterionic detergents.

### Claim 31 (depends on 29)

A method according to claim 29, wherein the lysis buffer further comprises one or more substances selected from the group consisting of chelating agents, reducing agents, inorganic salts pH indicators, and stabilizers.

### Claim 32 (depends on 31)

The method according to claim 31, wherein the lysis buffer has a pH in the range of from 4 to 11, from 7 to 10, or from 8 to 9.

### Claim 33 (depends on 16)

The method according to claim 16, wherein a proteolytic agent either is added to the mixture in step (3) in addition to the lysis buffer, or a proteolytic agent is already comprised in the lysis buffer added to the sample in step (3), said proteolytic agent being selected from the group consisting of proteases and non-enzymatic proteolytic compounds.

### Claim 34 (depends on 33)

The method according to claim 33, wherein said proteolytic agent is selected from the group consisting of proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys-C, bromocyane, recombinant Bacillus proteases, Lysozyme, and a mixture thereof.

### Claim 35 (depends on 16)

The method according to claim 16, wherein the step of incubating the mixture obtained after adding the aqueous lysis buffer to the sample according to step (4) is carried out at a temperature in the range of from 15° C. to 95° C.

### Claim 36 (depends on 35)

The method according to claim 35, wherein the step of incubating the mixture obtained after adding the aqueous lysis buffer to the sample according to step (4) is carried out at a temperature in the range of from 20° C. to 70° C.

### Claim 37 (depends on 36)

The method according to claim 36, wherein the step of incubating the mixture obtained after adding the aqueous lysis buffer to the sample according to step (4) is carried out at a temperature in the range of from 37° C. to 65° C.

### Claim 38 (depends on 16)

The method according to claim 16, wherein the step of incubating the mixture obtained after adding the aqueous lysis buffer to the sample according to step (4) is carried out at a temperature in the range of from 15° C. to 95° C. for 30 seconds to 24 hours, 50 seconds to 12 hours, 50 seconds to 5 hours, or 1 minute to 2 hours.

### Claim 39 (depends on 17)

The method according to claim 17, wherein the step of isolating and/or purifying the nucleic acid comprised in the aqueous phase according to step (6) is carried out by means of at least chromatographic and/or solid phase-based method.

### Claim 40 (depends on 39)

The method according to claim 39, wherein the at least chromatographic and/or solid phase-based method is selected from the group consisting of gel filtration chromatography, ion exchange chromatography, reversed phase chromatography, and chaotrope-free affinity binding.

### Claim 41 (depends on 39)

The method according to claim 39, wherein the lysis buffer comprises an anionic surfactant ion, and wherein after incubation according to step (4), the anionic surfactant ion is essentially removed from the sample by precipitation using monovalent ions of alkali metals and/or divalent ions of alkaline earth metals prior to isolating and/or purifying the nucleic acid from the aqueous phase.

### Claim 42 (depends on 41)

The method according to claim 41, wherein the monovalent ions of alkali metals and/or divalent ions of alkaline earth metals are selected from the group consisting of Rb+, Cs+, Ca2+, Sr2+, Ba2+, and a mixture thereof.

### Claim 43 (depends on 41)

The method according to claim 41, wherein isolating and/or purifying the nucleic acid from the aqueous phase is performed using gel filtration chromatography.

### Claim 44 (depends on 16)

The method according to claim 16, wherein the nucleic acids are selected from the group consisting of ribonucleic acids (RNA) and desoxyribonucleic acids (DNA).

### Claim 45 (depends on 16)

The method according to claim 16, wherein the nucleic acids are DNA.

### Claim 46 (depends on 16)

The method according to claim 16, wherein the nucleic acids are DNA having a length of at least 100 bp.

### Claim 47

A method for extracting, isolating, and/or purifying nucleic acids from a cross-linked wax-embedded sample, comprising:
lysing the cross-links in the sample in a bi- or multi-phasic mixture that comprises (a) one or more solvents selected from the group consisting of linear, branched, and cyclic C10-C16 alkanes and mixtures thereof, and (b) an aqueous lysis buffer.

### Claim 48 (depends on 47)

The method according to claim 47, wherein the cross-linked wax-embedded sample is a FFPE-sample.

### Claim 49 (depends on 47)

The method according to claim 47, wherein the one or more solvents are selected from the group consisting of C13-C16 alkanes and mixtures thereof.

### Claim 50 (depends on 47)

The method according to claim 47, wherein the one or more solvents are hexadecane.

### Claim 51 (depends on 16)

A kit for extracting, isolating and/or purifying nucleic acids from a wax-embedded sample according to the method of claim 16, comprising (a) a wax-solubilizing agent, and optionally (b) instructions for carrying out the lysis in a bi- or multi-phasic system according to step (3).

### Claim 52 (depends on 51)

The kit according to claim 51, further comprising one or more additional components selected from the group consisting of (c) an aqueous lysis buffer, (d) a proteolytic agent, and (e) a solid phase for chromatography and/or affinity binding.