# US 2014/0154776 A1 · Stable Lysis Buffer Mixture for Extracting Nucleic Acids

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- Number: US 2014/0154776 A1
- Document ID: US20140154776A1
- Status: Published application
- Kind: A1
- Application number: 14/171,157
- Filed: 2014-02-03
- Published: 2014-06-05
- Earliest claimed: 2008-04-22
- Assignees: STRATEC Biomedical AG
- Inventors: Peter Bendzko; Hans Joos
- Web page: https://patentgrep.com/patent/US20140154776A1

## CPC

- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 9/2462: {Lysozyme (3.2.1.17)}
- C12N 9/6424: {Serine endopeptidases (3.4.21)}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1013: {by using magnetic beads}
- C12N 15/1017: {by filtration, e.g. using filters, frits, membranes}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Embodiments relate to a lysis buffer mixture that is stable in storage for isolating nucleic acids from biological, preferably diagnostic samples. The mixture is preferably associated with an extraction control. The aim of the invention is to provide an improved nucleic acid extraction system, which is cost-effective, stable and easy to use, thus fulfilling the requirements of a modern nucleic acid extraction system and containing, among other things, extraction controls. Embodiments relate to a lysis buffer mixture for isolating nucleic acids, said mixture containing non chaotropic salts, a special selection of detergents, a defined quantity of at least one nucleic acid as an extraction control, optionally lytic enzymes, optionally carrier nucleic acids and optionally other additives.

## Claims

### Claim at position 1

1-28. (canceled)

### Claim 29

A method for preparing storage-stable, freeze-dried lysis buffer mixture for the isolation of nucleic acids from arbitrary complex initial substances, wherein said method comprises individual preparation of a first aqueous stem solution and a second aqueous stem solution, which are then mixed with one another, followed by mechanically portioning and freeze drying, wherein said first aqueous stem solution contains a mixture of at least one non-chaotropic salt, Tris, at least one detergent and at least one lytic enzyme, and wherein said second aqueous stem solution contains a nucleic acid for extraction control, carrier nucleic acids, sugar, and Tris-HCl.

### Claim 30 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the non-chaotropic salts of said first aqueous stem solution contain cations selected from the group consisting of monovalent cations, bivalent cations, multivalent cations, and mixtures thereof.

### Claim 31 (depends on 30)

The method of preparing the lysis buffer mixture of claim 30 wherein the cations are selected from the group consisting of ammonium ions, sodium ions, potassium ions, magnesium ions, calcium ions, zinc ions and manganese ions.

### Claim 32 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the pH value of the lysis buffer mixture is set between 5 and 9 when in aqueous solution prior to being freeze-dried.

### Claim 33 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29 wherein the at least one detergent of said first aqueous stem solution is selected from the group consisting of cetyltrimethylammonium bromide, Tween 20, Triton X-100, and sodium dodecyl sulfate.

### Claim 34 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the at least one detergent of said first aqueous stem solution is cetyltrimethylammonium bromide.

### Claim 35 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the at least one detergent of said first aqueous stem solution is present in the range of 0.1-5% of the total quantity.

### Claim 36 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein cetyltrimethylammonium bromide (CTAB) of said first aqueous stem solution is present as 2% of the total quantity.

### Claim 37 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the defined quantity of at least one nucleic acid as an extraction control of said second aqueous stem solution is selected from the group consisting of a synthetic nucleic acid, a nucleic acid manufactured via molecular biology methods, and a native nucleic acid.

### Claim 38 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the defined quantity of at least one nucleic acid for extraction control of said second aqueous stem solution is selected from the group consisting of at least one DNA, at least one RNA, and mixtures of at least one DNA and at least one RNA.

### Claim 39 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the at least one lytic enzymes of said first aqueous stem solution are proteinases.

### Claim 40 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the at least one lytic enzymes of said first aqueous stem solution are proteinase K.

### Claim 41 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, comprising lytic enzymes, wherein the at least one lytic enzymes of said first aqueous stem solution are proteinase K and lysozyme.

### Claim 42 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the carrier nucleic acids of said second aqueous stem solution are synthetic nucleic acids or isolates from biological materials.

### Claim 43 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the carrier nucleic acids of said second aqueous stem solution are poly A RNA, tRNA, salmon sperm DNA, or herring sperm DNA.

### Claim 44 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the lysis buffer mixture is prepared as a solid, storage-stable formulation in ready-to-use reaction vessels.

### Claim 45 (depends on 44)

The method of preparing the lysis buffer mixture of claim 44, wherein the ready-to-use reaction vessels are 96 well microtitre plates, 384 well microtitre plates, or 0.5-5 ml reaction vessels.

### Claim 46 (depends on 44)

The method of preparing the lysis buffer mixture of claim 44, wherein the ready-to-use reaction vessels are 2 ml reaction vessels.

### Claim 47 (depends on 29)

The method of preparing the lysis buffer mixture of claim 29, wherein the lysis buffer mixture is prepared in a tablet form.