EP2324115A1

EP2324115A1 - Method for isolating and purifying nucleic acids

Info

Publication number: EP2324115A1
Application number: EP09811122A
Authority: EP
Inventors: Ralf Himmelreich; Sabine Werner
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2008-09-03
Filing date: 2009-09-02
Publication date: 2011-05-25
Also published as: US8624020B2; WO2010026167A1; EP2163621A1; US20110224419A1

Abstract

The present invention relates to a method for isolating and purifying nucleic acids through elution of the nucleic acids from samples containing nucleic acids and from biological materials. The present invention further relates to a kit for carrying out the method according to the invention.

Description

Method for isolating and purifying nucleic acids

The present invention relates to a method for isolating and purifying nucleic acids by separating the nucleic acids of nucleated acidic samples, as well as biological materials. The present invention further relates to a kit for carrying out the method according to the invention.

A method for isolating and purifying nucleic acids, which is already known in the prior art, is based on the adsorption of nucleic acids on glass or silica particles in the presence of chaotropic salts, followed by the recovery of the adsorbed nucleic acids (Vogelstein, B. and Gillespie, D. (1979) "Preparative and analytical purification of DNA from Agarose", Proc Natl Acad, U.S.A. 76: 615-619.) According to this method, using high concentrations of chaotropic salts, such as, for example, sodium iodide, Sodium perchlorate or guanidinium thiocyanate, DNA isolated and purified by agarose The RNA or DNA can also be isolated or purified from various mixtures (Boom, R ₁ , 1990: "Rapid and Simple Method for Purification of Nucleic Acids", J. Clin. Microbiol. 28, 495-503).

After nucleic acid purification, nucleic acids are often used in the polymerase chain reaction (PCR). The PCR amplifies nucleic acids in a sequence-specific manner and is therefore widely used in gene or DNA diagnosis. The application of the PCR technique in routine clinical procedures poses several problems. It is known that inhibitory substances that have not been removed from the nucleic acid purification step can inhibit the PCR. Such inhibitory substances are, for example, hemoglobin and surfactants used in the nucleic acid extraction process. From this background, it can be seen that the methods for extracting and purifying nucleic acids are very important and relevant (Oshima et al., JJCL A, 22 (2) 145-150 (1997)).

Methods for extraction and purification of nucleic acids are very often automated. There are already automated nucleic acid extraction methods in the prior art, such as described in JP-A-107854/1999 and JP-A-266864/1999. In most procedures For the isolation and purification of nucleic acids, a solution which contains salts at high concentration and contains alcohol at a high concentration, and in which the nucleic acids are present, is brought into contact with an adsorption surface. The adsorption surface may be in the form of a resin in a column. Then, the nucleic acids are adsorbed to this surface and later eluted with the aid of solutions containing less highly concentrated saline solutions.

The problem with most nucleic acid isolation and purification procedures is that the yield of nucleic acids is relatively low. Another problem is that ethanol-containing solutions are classified as hazardous materials (HAZMAT) in accordance with IATA regulations (International Air Transport Association). According to the IATA regulations, all products, materials and goods are classified into nine main classes. Classification as dangerous goods will incur additional charges and taxes for air transport. It was therefore an object of the present invention to substantially replace ethanol (or isopropanol) in the process for purifying and extracting nucleic acids in order to facilitate the isolation and purification of nucleic acids, to provide an ethanol-free process and to facilitate air freight transport.

Substitutes for alcohol in processes for the purification of nucleic acids are known from the prior art, which, however, only partially solve the problems discussed above (US 2004/0167324). The majority of the substances described herein either fall into the HAZMAT IATA regulations or have a pungent odor that permits preparation only in the print.

Surprisingly, in the context of the present invention, solvents for wash buffers have been found for washing surface-immobilized nucleic acids which allow the wash buffer to be substantially free of alcohol, ie ethanol.

The present invention thus relates to a method for washing nucleic acids immobilized on surfaces, wherein nucleic acids immobilized on surfaces are brought into contact with a washing buffer and the washing buffer is substantially free from ethanol. Substantially free of etanianol in the context of the present invention means that ethanol is present in a concentration of less than 24% by volume, preferably less than 16% by volume and most preferably not at all in the wash buffer.

In particular, the present invention relates to a method for washing nucleic acids immobilized on surfaces, wherein the wash buffer contains solvents selected from the group comprising C3 to C5 alkyldiols, and short chain ethylene glycol derivatives and various water soluble polymeric compounds, wherein the wash buffer substantially is free of ethanol.

In a preferred embodiment, the present invention is a process for washing surface-immobilized nucleic acids, wherein the washing buffer contains solvents which are selected from the group comprising 1,2-butanediol, 1,2-propanediol, 1,3-butanediol, l -methoxy-2-propanol, 3-methyl ~ l, _3} 5-pentanetriol, DBE-2 dibasic ester, DBE dibasic ester-3, DBE-4 dibasic ester, DBE dibasic ester 5, DBE-6 dimethyl adipate, diethylene glycol monoethyl ether ( DGME), diethylene glycol monoethyl ether acetate (DGMEA), ethyl lactate, ethylene glycol, poly (2-ethyl-2-oxazoline), poly (4-styrenesulfonic acid-co-maleic acid) sodium salt solution, tetraethylene glycol (TEG), tetraglycol (tetrahydrofurfuryl polyethylene glycol ether), tetrahydrofurfuryl poly- ethylene glycol 200, tri (ethylene glycol) divinyl ether, anhydrous triethylene glycol, triethyl englycol monoethyl ether.

The washing buffer is particularly preferably selected from the group comprising tetraglycol, tetraethylene glycol, 1,3-butanediol, 1,2-butanediol and diethylene glycol monoethyl ether.

Furthermore, the subject matter of the present invention is a method for extracting nucleic acids from a solution comprising the steps:

(a) Adding a binding mediator to the nucleic acid-containing solution

(b) contacting the solution containing the binding mediator and the nucleic acids having a surface under chaotropic and / or high salt conditions

(c) binding or adsorption of the nucleic acids to the surface,

(d) washing the surface according to the method of the invention for washing surface-immobilized nucleic acids (e) recovering the nucleic acids bound or adsorbed to the surface by elution.

Chaotropic conditions are achieved by the addition of chaotropic substances. As chaotropic chemical substances are referred to dissolve the ordered hydrogen bonds in aqueous solutions. They thus reduce the hydrophobic effect and have a denaturing effect on proteins, since the driving force of protein folding is the assembly of the hydrophobic amino acids in the water. Examples of chaotropic substances are barium salts, guanidinium hydrochloride, thiocyanates such as guanidinium thiocyanate, perchlorates or sodium chloride. Chaotropic salts can be used according to their solubility product in concentration ranges between 1 M and 8 M.

High salt conditions means highly concentrated salt solutions, the concentration of salts in the solution being at least 1 M, preferably WM.

However, alternative measures to chaotropic or high-salt conditions can be made, which achieve the same effect, namely the binding of the nucleic acids to be purified to the surface.

Preferably, the binding mediator is selected from the group consisting of diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, furfuryl alcohol, poly (I -vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), poly (2-ethyl-2-oxazoline), poly (4-ammonium-styrenesulfonic acid) _J Tetraethylene glycol dimethyl ether, tetraethylene glycol, tetrahydrofurfuryl polyethylene glycol 200 and triethylene glycol monoethyl ether.

The person skilled in the art can also successfully replace ethanol in the binding process in the nucleic acid preparation by mixtures of said binding mediators. Since ethanol-containing solutions up to 24% (vol / vol) are not classified as HAZMAT, mixtures of the binding mediators can be used with ethanol.

The binding mediators are preferably present in the following concentrations;

Diethylene glycol monoethyl ether (DGME) [CAS 111-90-0] - Concentration range 70-99% by volume, preferred concentration 99.0%; in combination with ethanol; 60-80% by volume of DGME and 16-24% by volume of ethanol Diethylene glycol monoethyl ether acetate (DGMEA) [CAS 112-15-] -

Concentration range 70-99% by volume, preferred concentration 99.0% by volume; in

Combination with ethanol: 60-80Vol% DGMEA and lό-24Vol% ethanol furfuryl alcohol [CAS 98-00-] - Concentration range 20-30% by volume, preferred concentration 30% by volume

Poly (I -vinylpyrrolidone-co-2-dimethylammoethylmethacrylate) [CAS 30581-59-0] -

Concentration range 3 ~ 5Vol%, preferred concentration 5Vol% poly (2-ethyl-2-oxazoline) [CAS 25805-17-] - Concentration range 9-15Vol% (w / v), preferred concentration 12Vol%; in combination with ethanol: 22.5% (w / v) and 16-24% (v / v) ethanol

Poly (4-ammoniumstyrenesulfonic acid) [CAS 29965-34-] - Concentration range 8-

22% by volume (w / v), preferred concentration 12% by volume; in combination with ethanol: 8-

22 (w / v) and 24 vol% (v / v) ethanol

Tetraethylene glycol dimethyl ether [CAS 143-24-] - Concentration range 70-98% by volume, preferred concentration 98% by volume; in combination with ethanol: 73.5Vol% and

24Vol% ethanol tetraglycol (tetrahydrofurfuryl polyethylene glycol ether) [CAS 9004-76-] - preferred

Concentration with ethanol: 75% by volume and 16-24% by volume of ethanol Tetrahydrofurfurylpolyethylene glycol 200 [CAS 31692-85-0] - Concentration range 70-100% by volume, preferred concentration 100% by volume

Triethylene glycol monoethyl ether [CAS 112-50-5] - Concentration range 70-90% by volume, preferred concentration 90% by volume

Elution buffers are generally buffered low salt solutions with a neutral to slightly alkaline pH (eg buffer TE 10 mM Tris / Cl pH 8, 1 mM EDTA). The skilled artisan partly also uses distilled water,

In a particularly preferred variant of the method according to the invention, the washing buffer is tetraglycol in a concentration of 45% by volume to 80% by volume, preferably 52% by volume to 72% by volume; In the presence of 2 to 4 M, preferably 2.5 M of chaotrope, e.g. Guanidiniurnhydrochlorid is the preferred concentration of tetraglycol at 45 to 55 vol%, preferably at 50 vol%.

In another particularly preferred variant of the method according to the invention is the Washing buffer tetraethylene glycol in a concentration of 45 to 80 vol%, preferably from 50 vol% to 70 vol%. In the presence of 2 to 4 M, preferably 2.5 M of chaotrope, such as guanidinium hydrochloride, the preferred concentration of tetraethylene glycol is 45 to 55% by volume, preferably 50% by volume.

In another particularly preferred variant of the process according to the invention, the washing buffer is 1,3-butanediol in a concentration of 45 to 85% by volume, preferably of 51% to 80% by volume. In the presence of 2 to 4 M _3, preferably 2.5 M of chaotrope such as guanidinium hydrochloride, the preferred concentration of 1,3-butanediol is 45 to 55% by volume, preferably 49% by volume.

In another particularly preferred variant of the process according to the invention, the washing buffer is 1,2-butanediol in a concentration of 41% by volume to 71% by volume. In the presence of 2 to 4 M, preferably 2.5 M of chaotrope, e.g. Guanidinium hydrochloride is the preferred concentration of 1, 2-butanediol at 41 to 55Vol%, preferably 49Vol%.

In another particularly preferred variant of the process according to the invention, the washing buffer is present in a concentration of 38 to 98% by volume, preferably of 60% to 98% by volume. In the presence of 2 to 4 M, preferably 2.5 M of chaotrope, e.g. Guanidmium hydrochloride is the preferred concentration of 1, 2-butanediol at 38 to 45% by volume, preferably 42% by volume.

The surface to which the nucleic acids are adsorbed may be based on materials which may be selected from the group consisting of silica materials, carboxylated surfaces, zeolites, and titanium dioxide.

In the method according to the invention for the extraction of nucleic acids from a solution, the chaotropic and or high-salt conditions of reaction step b) are preferably achieved by the addition of chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride to the nucleic acid-containing solution.

The nucleic acid may be DNA such as genomic DNA. The nucleic acid may also be RNA, such as total RNA. The nucleic acid may be single-stranded or double-stranded nucleic acid, for example short double-stranded DNA fragments.

The nucleic acid-containing solution can be obtained by a lysis process from a nucleic acid-containing material.

The nucleic acid-containing material may be selected from the group consisting of blood, tissue, swabs, bacterial cultures, urine, cell suspensions and adherent cells, PCR reaction mixtures, and in vzYrσ nucleic acid modification reaction mixtures. The nucleic acid-containing material may include human, animal or plant material.

The nucleic acid-containing solution may be derived from a biochemical nucleic acid modification reaction.

It is preferred that surfactants are added to the nucleic acid-containing solution. These surfactants are preferably used in concentration ranges from 0.1% by volume to 10% by volume. Further, anti-foaming agents may be added, preferably in a range of 0.01 to 1% by weight.

The present invention also provides a reagent kit for washing surface-immobilized nucleic acids comprising a solution 1 comprising a washing buffer, wherein the washing buffer is substantially free of ethanol.

The present invention particularly relates to a reagent kit for washing nucleic acids immobilized on surfaces

a solution 1 comprising the wash buffer selected from the group consisting of C3- and C4-alkyldiols, as well as short-chain ethylene glycol derivatives and various water-soluble polymeric compounds and wherein the wash buffer is substantially free of ethanol.

In a preferred embodiment of the reagent kit for washing surface-immobilized nucleic acids, the reagent kit comprises a solution 1 comprising the wash buffer selected from the group consisting of 1, 2-butanediol, 1,2-propanediol, 1,3-butanediol, 1-methoxy-2-propanol acetate, 3-methyl

^" 1,3,5-pentanetriol, DBE-2 dibasic ester, DBE-3 dibasic ester, DBE-4 dibasic ester, DBE-5 dibasic ester, DBE-6 dimethyl adipate,"

Diethyl glycol monoethyl ether (D GME), diethylene glycol monoethyl ether acetate

(DGMEA), ethyl lactate, ethylene glycol, pory (2-ethyl-2-oxazolm), poly (4-styrenesulfonic acid-co-maleic acid) sodium salt solution, tetraethylene glycol (TEG),

Tetraglycol (tetrahydrofurfuryl polyethylene glycol ether), tetrahydrofurfuryl-pol y-ethylene glycol 200, tri (ethylene glycol) divinyl ether, anhydrous triethylene glycol,

Triethylene glycol monoethyl ether.

In a particularly preferred embodiment of the reagent kit for washing surface-immobilized nucleic acids, the reagent kit comprises

- A solution 1 comprising the wash buffer selected from the group comprising tetraglycol, tetraethylene glycol, 1,3-butanediol, 1, 2-butanediol and diethyl englycol monoethyl ether.

The present invention also provides a reagent kit for the extraction of nucleic acids from a solution comprising the abovementioned reagent kit and additionally comprising a mixture 2 comprising binding mediator, and

a mixture 3 comprising an eluent.

Preferably, the binding mediator is selected from the group comprising diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, furfuryl alcohol, poly (1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), poly (2-ethyl-2-oxazoline),

Poly (4-ammonium styrenesulfonic acid), tetraethylene glycol dimethyl ether, tetraethylene glycol, tetrahydrofurfuryl polyethylene glycol 200 and triemylene glycol monoethyl ether.

The person skilled in the art can also replace mixtures of the binding mediators mentioned successfully with ethaiol in the binding process in the nucleic acid preparation. Since ethanol-containing solutions can not be classified as HAZMAT up to 24% (vol / vol), mixtures can also be classified the binding mediators are used with ethanol.

The binding mediators are preferably present in the following concentrations:

Diethylene glycol monoethyl ether (DGME) [CAS 111-90-0] - Concentration range 70-99% by volume, preferred concentration 99.0% by volume; in combination with ethanol: 60-

80Vol% DGME and 16-24Vol% ethanol diethylene glycol monoethyl ether acetate (DGMEA) [CAS 112-15-] -

Concentration range 70-99% by volume, preferred concentration 99.0% by volume; in combination with ethanol: 60-80Vol% DGMEA and 16-24Vol% ethanol Furfurylalcohol [CAS 98-00-] - Concentration range 20-30Vol%, preferred

Concentration 30% by volume of poly (1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate) [CAS 30581-59-0] -

Concentration range 3-5Vol%, preferred concentration 5Vol% poly (2-ethyl-2-oxazoline) [CAS 25805-17-] - Concentration range 9-15Vol% (w / v), preferred concentration 12Vol%; in combination with ethanol: 22.5% (w / v) and 16-24% (v / v) ethanol

Poly (4-ammonium styrenesulfonic acid) [CAS 29965-34-] - Concentration range 8-22% by volume (w / v), preferred concentration 12% by volume; in combination with ethanol: 8-22 (w / v) and 24% (v / v) ethanol tetraethylene glycol dimethyl ether [CAS 143-24-] concentration range 70-98% by volume, preferred concentration 98% by volume; in combination with ethanol: 73.5% by volume and 24% by volume ethanol tetraglycol [CAS 9004-76] - preferred concentration with etlianol: 75% by volume and 16% by weight

24Vol% Ethanol Tetrahydrofurfurylpolyethylene glycol 200 [CAS 31692-85-0] - Concentration range

70-100 vol%, preferred concentration 100 vol%

Elution buffer are usually buffered low salt solutions with neutral to slightly alkaline pH (eg: buffer TE Fa. QIAGEN GmbH, Hilden). The skilled artisan sometimes also uses distilled water.

The present invention furthermore relates to a reagent kit for extracting nucleic acids from a solution comprising the abovementioned reagent kit and comprising a further solution 4 comprising a lysis buffer and a protease.

In the method according to the invention for the extraction of nucleic acids from a solution, the chaotropic and / or high-salt conditions of reaction step b) are, as already explained above, preferably by the addition of chaotropic salts such as potassium iodide, guanidine hydrochloride, guanidine thiocyanate or sodium chloride to the nucleic acid-containing solution reached.

D all is the subject of the present invention, a reagent kit according to the invention for the extraction of nucleic acids from a solution, wherein one of the solutions contains a chaotropic salt or high-salt conditions.

The chaotropic salt is preferably selected from a group comprising potassium iodide, guanidine hydrochloride, guanidine thiocyanate and sodium chloride,

The present invention also relates to the use of a reagent kit according to the invention for the extraction of nucleic acids from biological materials such as blood, tissue, smear preparations, bacteria, cell suspensions and adhering cells.

The present invention also provides the use of a reagent kit according to the invention for the purification of nucleic acids from biochemical reaction mixtures, PCR reaction mixtures and in vitro

Nucleic acid modification reaction mixtures.

The products, buffers and protocols described in the present application (process instructions) are published documents and commercially available products of QIAGEN GmbH, Hilden, Germany, unless otherwise indicated. Figure description

Fig. 1:

Behavior of tetraethylene glycol and tetraglycol used in QIAamp ^® spin 96 blood

protocol

Left: Normalized yields, determined by β-actin qPCR; right: agarose gel with the different samples Al: tetraethylene glycol

First Wash Buffer: WBl 0: 4S, 5Vol% tetraethylene glycol; 2.5 M GuHCl

Second wash buffer: WB6: 69.8% by volume tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH

7.5

Second wash buffer: WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

A2: tetraglycol

First wash buffer: WB 1 1: 50.0% by volume tetraglycol; 2.5 M GuHCl

Second wash buffer: WB6: 72.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Second wash buffer: WB 7: 52.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 2:

Behavior of 1,3-butanediol and 1,2-butanediol used in the QIAamp ^® 96 spin blood protocol Links: Normalized yield, as determined by ß-actin qPCR; right: agarose gel with the different samples

Al: 1,3-butanediol

First washing buffer: WB10: 49.0% by volume 1, 3-butanediol; 2.5 M GuHCl

Second wash buffer: WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Second wash buffer: WB7: 51.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH

7.5

A2: ^{'1,2-butanediol}

First washing buffer: WB 10: 49.0% by volume 1, 2-butanediol; 2.5 M GuHCl Second wash buffer: WB6: 71.0% by volume 1, 2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH

7.5

Fig. 3:

Behavior of diethylene used in QIAamp ^® 96 Spin Blood Protocol

Left: Normalized yields, determined by β-actin qPCR; right: agarose gel with the different samples First wash buffer: QIAGEN buffer AWl

First Wash Buffer: WB 11:42 by volume diethylene glycol monoethyl ether; 2.5 M GuHCl Second wash buffer: WB 1. 98% by volume diethylene glycol monoethyl ether Second wash buffer: WB6: 71% by volume diethylene glycol monoethyl ether; 10 mM NaCl;

Fig. 4:

Behavior of tetraethylene glycol and tetraglycol used in BioSprint ^® 96 DNA Blood protocol Links: Normalized yield, as determined by ß-aActin qPCR; right: agarose gel with the different samples

Al: tetraethylene glycol

First wash buffer: WB 10: 48.5% by volume tetraethylene glycol; 2.5 M GuHCl

Second wash buffer: WB6: 69.8 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Second wash buffer: WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH

7.5

A2: tetraglycol

First wash buffer: WB1 1: 50.0% by volume tetraglycol; 2.5 M GuHCl Second wash buffer: WB6: 72.0 vol% tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Second wash buffer: WB7: 52 ₃ 0Vol% tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 Fig S:

Behavior of 1,3-butanediol (Al), used in BioSprint ^® 96 DNA Blood protocol Links: Normalized yield, as determined by ß-actin qPCR; right: agarose gel with the different samples

First washing buffer: WB 10: 49% by volume 1,3 butanediol; 2.5M GuHCl Second wash buffer: WB2: 74 vol% 1.3 butanediol

Second wash buffer: WB5: 80% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 Second wash buffer: WB7: 51% by volume 1.3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 5:

Behavior of diethylene glycol monoethyl ether used in the BioSprint® 96 DNA Blood Protocol Left: Normalized yields determined by β-actin qPCR; right: agarose gel with the different samples

First wash buffer: QIAGEN buffer AWl

First Wash Buffer: WB 11: 42% by volume diethylene glycol monoethyl ether; 2.5 M GuHCl Second wash buffer: WB 1. 98% by volume diethylene glycol monoethyl ether Second wash buffer: WB6: 71% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig o:

QI Aquick® Purification of the Gel Pilot lkb ladder. Shown are the normalized

Exploit. The best wash buffers to replace the commercially available buffer PE contain 48-64% by volume tetraethylene glycol; 24% ethanol; 100 mM NaCl; 10 mM Tris pH 7.5

Fig. 7:

Behavior of 1,3-butanediol and 1,2-butanediol used in BioSprint ^® 96 Tissue

protocol

Left: Normalized yields, determined by Mouse-GapDH qPCR; right: agarose gel Al: 1,3-butanediol

First washing buffer: WB 10: 49.0 vol% 1, 3-butanediol; 2.5 M GuHCl

Second wash buffer: WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 Second wash buffer: WB7: 51, 0% vol 1, 3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

A2: 1,2-butanediol

First wash buffer: WBl 0: 49.0% by volume 1, 2-butanediol; 2.5 M GuHCl

Second wash buffer: WB6: 71.0% by volume 1, 2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 8:

Behavior of tetraethylene glycol and tetraglycol used in the BioSprint 96 Tissue Protocol

Left: Normalized yields, determined by Mouse-GapDH qPCR; right: agarose gel

Al: tetraethylene glycol

First wash buffer: WB10: 48.5% by volume tetraethylene glycol; 2.5 M GuHCl

A2: tetraglycol

Second wash buffer: WB7: 52.0 vol% tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Fig. 9:

Behavior of Diethyl engl ycolmonoetylyl etiler, used in the BioSprint 96 Tissue Protocol Left: Normalized yields, determined by Mouse-GapDH qPCR; right: agarose gel First Wash Buffer: WB 11: 42% by volume diethylene glycol monoethyl ether; 2.5 M GuHCl First Wash Buffer: Buffer AW1 Second Wash Buffer: WB 1: 98% by volume diethylene glycol monoethyl ether Second wash buffer: WB6; 71% by volume of diethylene glycol monoethyl ether; 10 mM NaCl; 10mM Tris-Cl pH 7.5

Fig. 10:

Behavior of tetraethylene glycol and tetraglycol used in DNeasy ^® 96 Tissue

protocol

Left: Normalized yields, determined by Mouse-GapDH qPCR; right: agarose gel

Al: tetraethylene glycol First Wash Buffer: WB 10: 48, 5% by volume tetraethylene glycol; 2.5 M GuHCl

Second wash buffer: WB7: 50.4 vol% tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 A2: tetraglycol

First wash buffer: WB 11: 50.0% by volume tetraglycol; 2.5 M GuHCl

Second wash buffer: WB6: 72.0% by volume tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Second wash buffer; WB7: 52.0% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 11:

Behavior of 1,3-butanediol and 1,2-butanediol used in BioSprint ^® 96 Tissue

protocol

First wash buffer: WB10: 49.0% by volume 1,3-butanediol; 2.5 M GuHCl

Second wash buffer: WB5: 80.0 vol% 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH

7.5

Second wash buffer: WB7: 51.0 vol% 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

A2: 1,2-butanediol

First washing buffer: WB10: 49.0% by volume of 1,2-butanediol; 2.5 M GuHCl

Second wash buffer: WB6: 71, 0% vol 1, 2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH

7.5 Fig. 12:

Behavior of tetraethylene glycol and 1,3-butanediol used in the RNeasy ^® 96 protocol Normalized Yields determined by Lamm RT- qPCR; used were "293" cells

Fig. L3:

Behavior of various ethanol-replacing chemicals used in the RNeasy ^® 96- Protocol

Left: Normalized yields, determined by Mouse-GapDH qPCR; right: agarose gel

additional bond

"1" 98Vol% TetraGIyme "2" 73.5Vol% TetraGIyme; 24% ethanol

Washing combination "a":

First wash buffer: 20% by volume tetraethylene glycol; 900mM GTC; 10 mM Tris / CL pH 7.5;

Second wash buffer: 70% by volume tetraethylene glycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Washing combination "b": First washing buffer: 20% by volume 1, 3-butanediol; 900mM GTC; 10 mM Tris / CL pH 7.5

Second wash buffer: 80% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 14:

Fragment size exclusion experiment

RNeasy® excludes small RNAs (5.8S, tRNA, miRNA, ...) during purification.

The limit size is about 150 bases. This experiment shows that the size exclusion of the tested chemicals is comparable to ethanol as a reference.

Wa: 20% by volume 1,3-butanediol; 900mM GTC; 10 mM Tris / CL pH 7.5 Wb: 60% by volume 1, 3 butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 Fig. 15:

Behavior of various ethanol-replacing chemicals used in the EpiTect Recovery Protocol 11. Left: Normalized yields, as determined by CFFI qPCR; right: "Delta Delta Ct" - analysis of different sample volumes. The calculation compares the measured Delta Ct values with the theoretical Delta Ct values, giving a numerical value for the degree of PCR inhibition.

B 90% by volume tetraethylene glycol; 1 OmM Tris-Cl pH 7.5 H 72% by volume 1, 3 butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

M 60% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Fig. 16:

cartridge assembly

Buffer ML mlD 4.5 M GTC; 50 mM NH 4 Cl; 45 mM Tris pH 7.5; 20 mM EDTA; 2.0 vol% Triton-X 100 ml 9 4.5 M GTC; 1.0 M NaCl; 50 mM NH 4 Cl; 45 mM Tris pH 7.5; 20 mM EDTA; 2.0Vol% Triton-X-100

MWl replacement buffer according to the present invention 2 49% by volume 1,3-butanediol; 2.5 mg / hl

MW2 replacement buffer according to the present invention

B 60% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig. 17:

Behavior of various ethanol-replacing chemicals used in EZl ^® DNA Blood 200 ul protocol

Top left: Normalized yields, determined by β-actin qPCR; right: "Delta Delta Ct" - analysis of different sample volumes. The calculation compares the measured Delta Ct values with the theoretical delta Ct values, giving a numerical value for the degree of PCR inhibition. Bottom left: agarose gel

Fig. 18:

Behavior of various ethanol-replacing chemicals used in the second

Washing step (after DNase digestion) of the EZ 1 ^® - RNA Protocol

Left: Cartridge arrangement; bottom left: Normalized yields, determined by MapK2

RT qPCR; bottom right: agarose gel MWl replacement buffer according to the present invention

"C" 56% by volume, 3-butanediol; 3 M GuHCl

"D-65" 65 vol% 1,3-butanediol; 1.75 M GuHCl

"D-55" 55% by volume 1,3-butanediol; 1.75 M GuHCl

"D ~ 50" 50% by volume 1,3-butanediol; 1.75 M GuHCl "D-45" 45% by volume 1, 3-butanediol; 1.75 M GuHCl

Buffer RPE Replacement buffer according to the present invention "2-60" 60% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-55" 55% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-50" 50% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 "2-45" 45% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Fig 19:

Behavior of different ethanol replacing chemicals used the EZl ^® in the first and second washing step - RNA Protocol

Above: Cartridge arrangement; left: Normalized yields, determined by MapK2 RT qPCR; bottom right: agarose gel

MWl replacement buffer according to the present invention "C 56% by volume 1,3-butanediol; 3 M GuHCl

"D-55" 55% by volume 1,3-butanediol; 1.75 M GuHCl

Buffer RPE replacement buffer according to the present invention

"2" 60% by volume 1, 3 butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

"2-50" 50% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5 Fig. 20:

Behavior of different ethanol replacing chemicals used the EZl ^® in the first and second Waschscliritt - RNA Protocol. Above: Cartridge arrangement; left: Normalized yields, determined by MapK2 RT qPCR; bottom right: agarose gel

Fig. 21:

Behavior of various ethanol-replacing chemicals used in the first and second wash cycles of the EZl-RNA protocol. Above: Cartridge arrangement; left: Normalized yields, determined by MapK2 RT qPCR; bottom right: agarose gel

The following examples are intended to explain the invention in more detail:

1. Chemicals and test kits / T estprotokolϊe with washing buffers according to the invention

1.1 Chemicals (summary listing):

1.2 test kits / test protocols

1.2.1 "BioSprint® 96 DNA Blood ¹

BioSprint ^® 96 with log file; "BS96_DNA_Blut_200"

Lysis β 200 μl blood »200 μl buffer AL

* 20 μl QIAGEN protease

* 15 min Incubate on a Thermomixer at 56 ° C and 1400 rpm binding β 200 ul of isopropanol to admit o 30 ul MagAttract ^® Suspension G (QIAGEN GmbH) to admit

washings

Reference protocol β Ix Buffer AWI (650 μl)

«Ix Buffer AWl (500 μl) «2x buffer AW2 (500 μl)

Buffer AW1 Replacement buffer according to the present invention

«50% by volume tetraglycol; 2.5M GuHCl * 48.5V% tetraethylene glycol; 2.5M GuHCl

• 49% by volume 1, 3-butanediol; 2.5M GuHCl

• 49% by volume 1,2-butanediol; 2.5M GuHCl

«42% by volume of diethylene glycol monoethyl ether; 2.5M GuHCl

Buffer AW2 replacement buffer according to the present invention

• 52% by volume tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

69.8 vol% tetraethylene glycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

80% by volume 1, 3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

71% by volume of 1,2-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 »71% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 1mM Tris

Cl pH 7.5

Water rinse: 0.02% by volume Tween 20

elution; 200 μl μl of RNase-free water in MicroTubePacl-MicroPlate

1.2.2 QIAamp ^® 96 Spin Blood Protocol

Lyse

• 200 μl blood • 200 μl buffer AL

• 20 ul QIAGEN Protease "mix β incubation for 15 min at 56 ° C β binding add 200 ul of ethanol in S block and transfer in QIAamp ^® 96 plate

Wash solutions • Reference method o Ix Buffer AWI (650 μl) o Ix Buffer AW2 (500 μl) Buffer AWl replacement solutions according to the present invention

• 49% by volume 1, 3-butanediol; 2.5M GuHCl

• 49% by volume of 1,2-butanediol; 2.5M GuHCl β 42% by volume diethyleglycol monoethyl ether; 2.5M GuHCl

• 50% by volume tetraglycol; 2.5M GuHCl

«48.5% by volume of tetraethylene glycol; 2.5M GuHCl

Buffer AW2 replacement solutions according to the present invention o 80% by volume of 1,3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

* 71% by volume 1,2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

* 71% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 10 mM Tris-Cl pH 7.6

* 52% by volume tetraglycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 • 50.4 vol% tetraethylene glycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Elution: 200 μl μl of RNase-free water in Elution Microtube Rack

1.2.3 "BioSprint ^® 96 DNA Tissue"

BϊoSprint ^® -96 log file; "BS96_DNA_Blut_200"

Lyse

200 μl lysate (25 mg tissue + 180 μl buffer ATL + 20 μl proteinase K, overnight incubation 56 ° C)

• + 200 μl buffer AE binding

«Add 200 μl isopropanol β + 30 μl MagAttract ^® Suspension G wash solutions o Ix Buffer AWI (650 μl) o Ix Buffer AWI (500 μl) o 2x buffer AW2 (500 μl) Buffer AWl replacement solutions according to the present invention

* 48.5% by volume tetraethylene glycol; 2.5M GuHCl

• 50% by volume 1,2-butanediol; 2.5M GuHCl "50Vol% tetraglycol; 2.5M GuHCl • 49Vol% l, 3-butanediol; 2.5M GuHCl

* 42% by volume diethylene glycol monoethyl ether; 2.5M GuHCl buffer AW2 replacement solutions according to the present invention β 50.4 vol% tetraethylene glycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

«52Vol% 1, 2-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 • 52% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

80% by volume 1, 3-butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 β 98% by volume diethylene glycol monoethyl ether, pure solution

Water rinse: 0.02% by volume Tween® 20 (500 μl) Elution: 200 μl of RNase-free water in microtube plate

1.2.4 "DNeasy ^® 96 Tissue"

Lysis «200 μl lysate (25 mg tissue + 180 μl buffer ATL + 20 μl proteinase K, sup

Incubate night 56 ⁰ C)

• + 200 μl buffer AE binding

»Add 200 μl of ethanol β in the S block and transfer to QIAamp ^® 96 plate

washings

• Ix Buffer AWI (650 μl)

• 1 x buffer AW2 (500 μl)

Buffer AWl replacement solutions according to the present invention

48.5% by volume tetraethylene glycol; 2.5M GuHCl β 49Vol% 1,2-butanediol; 2.5M GuHCl β 49Vol% 1,3-butanediol; 2.5M GuHCl β 50% by volume tetraglycol; 2.5M GuHCl Buffer AW2 replacement solutions according to the present invention

69.8 vol% tetraethylene glycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

71% by volume of 1,2-butanediol; 1 normal NaCl; 1 OmM Tris-Cl pH 7.5 "80% by volume 1,3-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

"52% by volume tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Elution: 200 μl of RNase-free water in Elution Micro Tubes

1.2.5 RNeasy ^® 96

binding

• 350 μl buffer RLT lysate ("293" cells, 2x10 ⁵ cells / sample) "Add 350 μl ethanol * mix in S block and transfer to RNeasy ^® 96 plate

washings

• 2x buffer RWl (650 μl)

• 2x Buffer RPE (500 μl)

Buffer RWl replacement solutions according to the present invention

«20Vol% 1, 3-butanediol; 900mM GTC; 10 mM Tris / CL pH 7.5

• 20% by volume tetraethylene glycol; 900mM GTC; 10 mM Tris / CL pH 7.5 buffer RPE replacement solutions according to the present invention β 80% by volume 1,3 butanediol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5 β 60% by volume tetraethylene glycol; 10 mM NaCl; 1 OmM Tris-Cl pH 7.5

Elution: 100 μl of RNase-free water in Elution Micro Tubes

1.2.6 QIAquick ^®

binding

- * 1 volume of sample β 5 volumes of buffer PM admit (standard reference protocol) β on QIAamp MinElute ^® column load; I ¹ @ 8000rpm wash solution

• 1 x wash solution: A; 1 '@ 8000rρm

Buffer PM Replacement Solution According to the Present Invention • 64% by volume tetraethylene glycol; 24% ethanol; 100 mM NaCl; 10mM «Centrifuge dry

• Elute 40μl RNase-free water; I ¹ @ 8000rpm

1.2.7 Not Dangerous Goods - Modified EpiTect ^® Bisulfite Protocol

1) Bisulfite-DNA conversion according to manual EpiTect ^® Bisulfite-Kit from QIAGEN GmbH

2) Purification of Bisulfite-converted DNA Step #:

6. Transfer the complete bisulfite reactions to clean 1.5 ml microcentrifuge tubes. Precipitants in the solution do not affect the performance or yield of the reaction.

7. Add 560 μl of freshly prepared buffer BL containing 10 μg / ml of carrier RNA. Mix the solution by vortexing.

Note: Carrier RNA is not required when using> 100 ng of DNA.

8. Transfer the entire mixture to the EpiTect® spin column.

9. Centrifuge the column at maximum speed for 1 min. Discard the flow and return the ejector column to the collection vessel. 10. Add 500 μl buffer BW (wash buffer) to the spin column and at

Centrifuge maximum speed for 1 min. Discard the flow and return the ejector column to the collection vessel.

11. Add 500 μl buffer BD (desulfonation buffer) to the spin column and incubate for 15 min at room temperature. If precipitates are present in buffer BD, avoid transferring them to the spin column. Note: It is important that

Close the column lid before incubation.

12. Centrifuge the column at maximum speed for 1 min. Discard the flow and place the ejector column back into the collection vessel. 13. Add 500 μl buffer BW and centrifuge for 1 min at maximum speed. Discard the flow and place the ejector column back into the receptacle.

14. Repeat step 13 once.

15. Place the spin column in a new 2 ml collection vessel and centrifuge the spin column at maximum speed for 1 min to remove any remaining fluid. Note: If the purified DNA is intended for use in a real-time PCR, increase the centrifugation time to 5 min.

16. Place the spin column in a clean 1.5 ml microcentrifuge tube. Add 20 μl buffer EB to the middle of the membrane. Purify the purified DNA by centrifugation for 1 min at approximately 15,000 x g (12,000 rpm).

Buffer BW replacement solutions according to the present invention for protocol step 13 "60% by volume diethylene glycol monoethyl ether; 10 mM NaCl; 1 OmM Tris-Cl pH 7-5 • 72% by volume 1,3 butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

1.2.8 EZ1 ^® DNA Blood 200uI-ProtokolI EZ1 Cartridge allocation schedule (til) Change position

^ 1.2.9 EZr - RNA protocol

EZ1 Cardiofile Assignment Plan (ul) I Interchangeable position + 100 "+456 * -, +640"

+785 «

2. Result examples

The results of the test kits / test protocols according to the invention are shown in FIGS. 1-21.

The studies presented here focused on the ethanol-replacing chemicals in wash buffers for the silica-mediated preparation of nucleic acids. The sample material tested ranged from blood to tissues as well as nucleic acid modification reaction-upregulation products. In addition, the purification of various types of nucleic acids was tested (genomic DNA, total RNA, small double-stranded DNA fragments). The identified chemicals were tested by methods using columns with silica-based membranes or silica-coated magnetic particles. Chemical CAS

Concentration / Buffer Compositions of Replacement Solutions Used in the Examples According to the Present Invention

1,2-butanediol [1,2-butanediol] 584-03-2

49% by volume 1,2-butanediol; 2.5M GuHCl 71Vol% 1,2-butanediol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

1,2-propanediol 57-55-6

99% by volume 1,2-propanediol 74% by volume 1, 2-propanediol, 10 μm NaCl; 1 OmM Tris-Cl pH 7.5

1,3-butanediol [1,3-butanediol] 107-88-0

49% by volume of 1,3-butanediol; 2.5M GuHCl 56Vol% 1,2-butanediol; 3 M GuHCl

20% by volume 1,3-butanediol; 900mM GTC; 10 mM Tris / CLpH 7.5

72-80% by volume 1,3-butanediol; 30-10 μm NaCl; 10 mM Tris-Cl pH 7.5

1-Methoxy-2-propanol acetate [2-methoxy-1-methylethyl acetate] 108-65-6

100% by volume of 1-methoxy-2-propanol acetate

3-methyl-1,3,5-pentanetriol 7564-64-9 80-40vol% 3-methyl-1,3,5-pentanetriol

60% by volume of 3-methyl-1,3,5-pentanetriol, 10 mM of NaCl; 10 mM Tris-Cl pH 7.5

DBE-2 dibasic ester [dibasic ester] MFCD00191969 100-50 vol% DBE-2

DBE-3 dibasic ester [dibasic ester] MFCD00191969 100-50% by volume DBE-3

DBE-4 dibasic ester [dibasic ester; Dimethlylic succinate] 106-65-0

100-50 vol% DBE-4

DBE-5 dibasic ester [dibasic ester; dimethyl]

1119-40-0

100-50 vol% DBE-5

DBE-6 dimethyl adipate [dibasic ester; Dimethyl adipate] 627-93-0 100-50% by volume DBE-5

Diethylene glycol monoethyl ether (DGME) [diethylene glycol monoethyl ether, ethyl diglycol] 111-90-0

42% by volume of DGME; 2.5M GuHCl

71% by volume of DGME; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Diethylene glycol monoethyl ether acetate (DGMEA) [ethyl diglycol acetate] 112-15-2

99.0% by volume of DGMEA

50-80% by volume of DGMEA; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Ethyl lactate [ethyl lactate] 97-64-3

100% by volume ethyl lactate

75% by volume of ethyl lactate, 10 mM of NaCl; 10 mM Tris-Cl pH 7.5

Ethylene glycol [ethyl englycol] 107-21-1

100 vol% ethylene glycol

Poly (2-ethyl-2-oxazolines) [2-ethyl-4,5-dihydro-1,3-oxazol] 25805-17-8

23% by volume poly (2-ethyl-2-oxazoline), 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Poly (4-styrenesulfonic acid-co-maleic acid) 68037-40-1 25% by volume poly (4-styrenesulfonic acid-co-maleic acid) 19% by volume of poly (4-styrenesulfonic acid-co-maleic acid) 10M NaCl; 10 mM Tris-Cl pH 7.5

Tetraethylene glycol (TEG) [tetraethylene glycol] 112-60-7

48 ₃ 5VoI% TEG; 2.5M GuHCl

20-65% by volume TEG; 900mM GTC; 10 mM Tris / Cl pH 7.5

50.4-60% by volume TEG; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

64% by volume TEG, 24% by volume ethanol; 50% by volume TEG, 50% by volume poly (propylene glycol), MW -725 (CAS 25322-69-4)

Tetraglycol 9004-76-6

50% by volume tetraglycol; 2.5M GuHCl 52Vol% tetraglycol; 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Tetrahydrofurfurylpol yethylglycol 200 31692-85-0 100% by volume (pur.) 75% by vol. Tetrahydrofurfuryl PEG 200, 10 μM NaCl; 1 OmM Tris-Cl pH 7.5

Tri (ethylene glycol) divinyl ether [triethylene glycol divinyl ether]

765-12-8

75-98% by volume of tri (ethylene glycol) divinyl ether Triethylene glycol anhydrous [triethylene glycol, anhydrous]

112-27-6

97% by volume triethylene glycol, anhydrous,

75% by volume triethylene glycol, anhydrous, 10 mM NaCl; 10 mM Tris-Cl pH 7.5

Triethylene glycol monoethyl ether [triethylene glycol monoethyl ether]

112-50-5

90% by volume triethylene glycol monoetliyletlier,

68% by volume triethylene glycol monoethyl ether, 10 μm NaCl; 10 mM Tris-Cl pH 7.5

Table 1 commercially available products from. QIAGEN, used in the examples

Claims

A method of washing surface-immobilized nucleic acids wherein the nucleic acids are contacted with a wash buffer containing less than 24% by volume of 1-3 C-C alcohols and at least one further solvent selected from alkanediols and Moles having 2-6 C atoms, monocarboxylic acid esters and dicarboxylic acid diesters having 2-6 C atoms in the acid moiety and 1-4 C atoms in the alcohol moiety; (Poly) ethylene glycols and ether and ester derivatives thereof, wherein "poly" denotes 2-200 ethylene glycol repeating units; poly (2-ethyl-2-oxazoline); poly (4-styrenesulfonic acid-co-malic acid) sodium salt solution.

2. A method for washing surface-immobilized nucleic acids according to claim 1 or 2, wherein the washing buffer contains solvents which are selected from the group comprising 1, 2-butanediol, 1,2-propanediol, 1,3-butanediol, 1-methoxy 2-propanol acetate, 3-methyl-l, 3,5-pentanetriol, DBE-2, DBE-3, DBE-4, DBE-5, DBE-6, diethylene glycol monoethyl ether (DGME), diethylene glycol monoethyl ether acetate (DGMEA), ethyl lactate, Ethylene glycol, poly (2-etyl-2-oxazoline), poly (4-styrene-co-maleic acid) sodium salt solution, tetraethylene glycol (TEG), tetraglycol, tetrahydrofurfuryl polyethylene glycol 200. tri (ethylene glycol) divinyl ether, triethylene glycol anhydrous, Triethylenglykolmonoetliylether.

3. Verfaliren for washing surface-immobilized nucleic acids according to any one of claims 1 to 3, wherein the washing buffer contains solvents which are selected from the group containing tetraglycol, tetraethylene glycol, 1,3-butanediol, 1, 2-butanediol and triethylene glycol monoethyl ether.

A method of extracting nucleic acids from a solution comprising the steps of: (a) adding a binding mediator to the nucleic acid-containing solution

(b) contacting the solution with the binding mediator and the nucleic acid-containing solution having a surface under chaotropic and / or high salt conditions

(c) binding or adsorption of the nucleic acids to a surface, (d) washing the surface according to a method according to any one of claims 1 to 4.

(e) recovering the nucleic acids adsorbed to the surface by elution.

5. The method according to any one of claims 1-4, wherein the nucleic acid is genomic DNA.

6. The method according to any one of claims 1-4, wherein the nucleic acid is total RNA.

7. The method according to any one of claims 1-4, wherein the nucleic acids are short double-stranded DNA fragments.

8. Reagent kit for washing nucleic acids immobilized on surfaces comprising

a solution 1 comprising a wash buffer, wherein the wash buffer is as defined in claims 1-4.

9. Reagent kit according to claim 8 additionally comprising

a solution 2 comprising binding mediator, and

a solution 3 comprising an eluent.

10. reagent kit according to claim 9, comprising a further solution 4 comprising a lysis buffer and a protease.

11. Use of a reagent kit according to any one of claims 8 to 10 for the extraction of nucleic acids from biological materials such as blood, tissue, smear preparations, bacteria, cell suspensions and adheri he ends cells.

12. Use according to claim 11 for the purification of nucleic acids from biochemical reactions, PCR reactions and in vitro nucleic acid modification reactions.