EP1299531A2

EP1299531A2 - Method for isolating nucleic acids

Info

Publication number: EP1299531A2
Application number: EP01971766A
Authority: EP
Inventors: Martin Weber; Thorsten Singer; Sarah Cosaert
Original assignee: Qiagen GmbH
Current assignee: Qiagen GmbH
Priority date: 2000-07-12
Filing date: 2001-07-12
Publication date: 2003-04-09
Also published as: WO2002004620A2; JP2004502458A; DE10033991A1; WO2002004620A3; US20040091875A1

Abstract

The invention relates to a method for isolating nucleic acids from a solution, wherein the nucleic acids are adsorbed on a surface containing SiO2 in the presence of alkali halides and alcohol. The invention also relates to the use of a buffer solution containing alkali halides for isolating nucleic acids on a carrier containing SiO2, in addition to a kit for implementing a method for isolating nucleic acids from a solution.

Description

Process for the isolation of nucleic acids

The present invention relates to a method for isolating nucleic acids from a solution, the nucleic acids being adsorbed on a Si0 ₂ -containing surface.

Furthermore, the present invention relates to the use of a buffer solution for isolating nucleic acids on a support containing SiO ₂ and to a kit for carrying out a method for isolating nucleic acids from a solution.

The purification and isolation of nucleic acids on mineral supports in the presence of chaotropic salts is well known in the literature.

Marko et al. [Analyte. Biochem. 121 (1982) 382] and Vogelstein et al. [Proc. Nat. Acad. Be. 76 (1979) 615] recognized that if the DNA from nucleic acid-containing extracts is exposed to high concentrations of sodium iodide or sodium perchlorate, only the DNA binds to mechanically finely crushed glass scintillation tubes and crushed glass fiber membranes or glass fiber plates, whereas RNA and proteins do not tie. The DNA bound in this way can optionally be eluted with water.

EP 0389063 B1 relates to a method for isolating nucleic acids from a biological source. According to this method, the biological sources containing nucleic acids, such as blood, cells, plasma, etc., are disrupted in high concentrations in the presence of chaotropic salts, and the nucleic acids are then attached bound a silica surface. These are then washed and eluted.

The method described in US Pat. No. 5,155,018 describes the isolation of RNA from biological sources which, in addition to RNA, also contain DNA and other ingredients.

The biological sample is acidified and treated with a chaotropic agent, e.g. mixed with a guanidinium salt. Silicate particles are added to the sample and, under the given conditions, RNA binds to the silicate particles. Subsequently, the RNA is also separated from the particles here.

Colpan et al. in WO 95/01359 disclose a process for the purification and separation of nucleic acid mixtures by adsorption of the nucleic acid from an alcohol-containing solution with a high ionic strength. In addition to alcohol in a concentration of 1 to 50% by volume, the adsorption solution contains salts in a concentration of 1 to 10 M, the chaotropic salts, e.g. Guanidinium thiocyanate, sodium perchlorate, or guanidinium hydrochloride are preferred.

WO 95/21849 relates to a method for separating double-stranded and / or single-stranded nucleic acids from sources which contain these nucleic acids. In this method, too, the nucleic acids are adsorbed on mineral carriers under conditions which allow binding of the desired nucleic acid species, while the undesired nucleic acid species does not bind on this mineral carrier.

In order to bind predominantly single-stranded nucleic acid to a mineral carrier and thus separate it from double-stranded nucleic acid, the treatment conditions for the two Samples containing nucleic acid species with an aqueous mixture of salts, in particular chaotropic salts, and alcohol adjusted accordingly. The non-adsorbed double-stranded nucleic acid can then be further purified or isolated using known methods.

Nucleic acids that are used for molecular biological applications, e.g. for PCR, sequencing, and gene transfer, in particular transfection or vaccination, are subject to very high requirements in terms of purity and integrity. The use of nucleic acids in molecular diagnostics or molecular medicine presupposes that they must be free of toxic substances, which can lead, for example, to pathogenic effects in the organisms to be treated.

The methods known from the prior art have in common that chaotropic salts are used in high concentrations for the isolation of nucleic acids on silica surfaces. Chaotropic substances such as guanidinium hydrochloride, guanidinium thiocyanate or sodium perchlorate are highly toxic substances. It cannot be ruled out that the nucleic acids isolated in the presence of these substances will be contaminated with them and thus cannot be used or can only be used to a limited extent for use in molecular biological applications.

The handling of chaotropic substances also represents a high health risk for the user, so that the handling of these substances must be carried out under certain protective measures.

The technical problem on which the present invention is based is to provide an improved method which uses known disadvantages overcome. The substances used in this method for binding the nucleic acid should not be toxic. The method should be as inexpensive as possible, for example by using inexpensive chemicals, and the isolated nucleic acids should be isolated quantitatively and qualitatively purely.

Surprisingly, the present invention provides a method for isolating nucleic acids from a solution which achieves this object. The invention consists in the fact that in a first step the binding of nucleic acids to Si0 ₂ -containing surfaces in the presence of alkali halides in a concentration of 0.1-3M, preferably 0.25-1.5M, and alcohol in a concentration from 37 to 70 vol.% is carried out. The nucleic acids adsorbed on the Si0 ₂ -containing surface are then optionally washed with an alcohol-containing washing buffer and the nucleic acid is eluted with an aqueous salt solution or with water.

Aqueous adsorption solutions are used to bind the nucleic acids to the Si0 ₂ -containing surface, the alkali halides such as NaCl, KCl and LiCl in a concentration of 0.1-3M, preferably 0.25-1.5M, particularly preferably 0.5 - 1, 25 M and in particular 0.5 - 1, 0 M included. Alkali halides are non-toxic substances and handling the salt solutions in the concentration used is harmless to health.

In addition to the salts mentioned, the aqueous adsorption solutions contain lower aliphatic, branched or unbranched alcohols with a chain length of 1 to 5 carbon atoms. The aliphatic alcohols present in the solution are preferably methanol, ethanol, propanol, isopropanol and butanol in a concentration of 37-70% by volume, preferably 37-50% by volume. Among the alcohols mentioned above, ethanol and / or isopropanol in a concentration of 37-70% by volume are particularly preferred.

Si0 ₂ -containing surfaces can, for example, be porous or non-porous silicon oxides or metal-silicon mixed oxides, silica gels, materials

Base of glasses, e.g. modified or unmodified glass particles or glass powder, quartz, zeolites or mixtures of one or more of the substances mentioned above.

For the purposes of the present invention, a surface is understood to mean any microporous separating layer.

In a particularly preferred embodiment of the invention

The Si0 ₂ -containing surface is a porous membrane or a process

Filters made of silica gel, glass or quartz fibers.

In a further embodiment of the method according to the invention, the term surface in the broader sense also includes a layer of particles or also a granulate and also fibers, such as, for. B. silica gel nonwovens.

According to the invention, water or aqueous salt solutions are suitable as eluents for eluting the bound nucleic acid. Buffer solutions known from the prior art are used as salt solutions, such as, for example, morpholinopropanesulfonic acid (MOPS), tris (hydroxymethyl) aminomethane (TRIS), 2- [4- (2-hydroxyethyl) -1-piperazino] ethanesulfonic acid (HEPES) in a concentration of 0.001 to 0.5 mol / liter, preferably 0.01 to 0.2 mol / liter, particularly preferably 0.01 to 0.05 molar solutions.

In a further embodiment of the method according to the invention, the nucleic acids in the eluate can preferably be isolated by alcoholic precipitation. The nucleic acids isolated with this method are free of toxic substances and are therefore suitable for use in molecular biology. In the following, the term "nucleic acid" is to be understood in its broadest sense, that is to say ribonucleic acids (RNA) as well as deoxyribonucleic acids (DNA) in all lengths and configurations, such as double-stranded, single-stranded, circular and linear, branched, etc. and all possible subspecies, such as monomeric nucleotides, oligomers, plasmids, cosmids, viral and bacterial DNA and RNA, as well as genomic and non-genomic DNA and RNA from animal and plant cells or other eukaryotes, mRNA in processed and unprocessed form, tRNA, hn-RNA, rRNA, cDNA and include all other conceivable nucleic acids.

The method according to the invention allows nucleic acids of any origin to be isolated from solutions. The sample containing nucleic acids comes, for example, from animal or vegetable tissues, tissue or cell cultures, bone marrow, human and animal body fluids such as blood, serum, plasma, urine, sperm, cerebrospinal fluid, sputum and smears, plants, plant parts and extracts, for example Juices, mushrooms, prokaryotic or eukaryotic microorganisms such as bacteria or yeast, fossil or mummified samples, soil samples, sewage sludge, waste water and food (especially processed, ie technically processed food). Nucleic acids which have arisen from chemical reactions, such as, for example, those which have been obtained by polymerase chain reaction (PCR) or plasmid DNA, genomic DNA and RNA and / or nucleic acids which come from microorganisms, can also be isolated according to the invention. The method according to the invention is particularly suitable for isolating plasmid DNA from bacteria, such as, for example, E. coli, for subsequent cloning, transfection or sequencing.

The bacteria are lysed using known lysis methods, such as alkaline lysis according to Bimboim and Doly (1979) or lysis by heating according to Holmes and Quigley.

The cell debris as well as the precipitated proteins and the genomic DNA are removed from the viscous lysate by centrifugation or filtration, and a clarified lysate is obtained which contains the plasmid DNA. The plasmid DNA can be purified, for example, by means of ion exchange chromatography, and the plasmid DNA pre-purified in this way can then be isolated using the method according to the invention.

Another object of the present invention is a kit for isolating nucleic acids from a solution, comprising a) an adsorption solution containing 0.25-1.5 M NaCl, KCI or a mixture thereof and ethanol or isopropanol in one

Concentration of 37 - 70 vol%; and b) a Si0 ₂ -containing surface.

The Si0 ₂ -containing surface can be a porous membrane or a filter made of silica gel, glass or quartz fibers and arranged in a suitable device. The kit preferably also contains solutions suitable for lysis and, as described above, washing and elution buffers.

The nucleic acids isolated according to the invention are free from ^; nucleic acid degrading enzymes and have such a high purity, that they can be processed and processed immediately in a variety of ways.

The nucleic acids produced according to the invention can be used for cloning and serve as substrates for a wide variety of enzymes, such as, for example, DNA polymerases, DNA restriction enzymes, DNA ligase and reverse transcriptase.

The nucleic acids provided by the method according to the invention are particularly well suited for amplification, in particular for PCR, strand displacement amplification, rolling circle methods, ligase chain reaction (LCR) and similar methods.

The method according to the invention is furthermore particularly suitable for providing nucleic acids for use in diagnostics, in particular for a diagnostic method, which is characterized in that the nucleic acid purified by the method according to the invention is amplified in a subsequent step and subsequently and / or simultaneously the nucleic acid amplified in this way is detected (e.g. Holland, PM et al., 1991. Proc. Natl. Acad. Sci. 88, 7276-7280. Livak, KJ et al., 1995. PCR Methods Applic. 4, 357 - 362; Kievits, T. et al., 1991. J. Virol. Meth. 35, 273-286; Uyttendaele, M. et al., 1994. J. Appl. Bacteriol. 77, 694-701).

Example 1 :

Isolation of plasmid DNA in the presence of NaCl and alcohol in various concentrations.

390 μl each of the individual buffers (0.25-1.5 M NaCl; 50 mM Tris, pH 8.5; 15% (w / v) isopropanol) were mixed with 10 μg plasmid DNA (pCMVß; Fa.

Clontech # 6177-1) (c = 1 μg / μf) mixed and with different amounts added to isopropanol, which corresponds to a total of 28.9-49.8 vol% isopropanol in the respective binding buffers. After five minutes of incubation at room temperature (20-25 ° C.), the batches were transferred to a column containing a silica membrane and passed through the silica membrane under vacuum (approx. 600 mbar; use of the QIAvac 6S from QIAGEN GmbH). The mixture was then washed with 750 μl buffer PE (10 mM Tris, pH 7.5; 80% ethanol) and drawn through the membrane until the air was dry. Elution was carried out by adding 100 μl of buffer EB (10 mM Tris, pH 8.5) and the yield was determined photometrically at 260 nm. The result is shown in Table 1.

Table 1 :

Dilution factor 20; Total volume 50 μl; Yield DNA 10 μg = 100% Example 2:

Isolation of plasmid DNA at different alcohol concentrations 500 μg (c = 1 μg / μl) plasmid DNA (pCMVß; Clontech # 6177-1) in 5 ml buffer Q1 (1, 25 M NaCl; 50 mM Tris, pH 8.5; 15 vol% isopropanol). Then isopropanol was added, in an amount corresponding to a total of 22.7-43.3 vol% isopropanol in the binding buffer, mixed thoroughly and incubated for 5 min at room temperature (20-25 ° C). The DNA / Q1 / isopropanol mixture was pressed into a glass fiber filter (Sartorius, Minisart series) and the membranes were blown out to dryness. Elution was carried out with 1 ml TE buffer (10 mM Tris, pH 7.5; 1 mM EDTA). The yield was determined photometrically at 260 nm. The result is summarized in Table 2.

Dilution factor: 20; Total volume: 1 ml Example 3:

Isolation of plasmid DNA in the presence of KCI and alcohol in various concentrations. 390 .mu.l of each buffer (0.25-1.0 M KCI; 50 mM Tris, pH 8.5; 15% (w / v) isopropanol) ) with 10 μg plasmid DNA (pCMVß; Clontech # 6177-1) (c = 1 μg / μl) and mixed with different amounts of isopropanol, which contain up to 57.3 vol% isopropanol (see table 3) or up to 71.4% by volume of isopronal together with ethanol in the respective binding buffers. After five minutes of incubation at room temperature (20 ° -25 ° C.), the batches were transferred to a column containing a silica membrane (QIAquick, QIAGEN GmbH, # 28104) and under vacuum (approx. 600 mbar; use of the QIAvac 6S from QIAGEN GmbH) through the silica membrane. The mixture was then washed with 750 μl of buffer PE (10 mM Tris, pH 7.5; 80% ethanol) and drawn through the membrane until the air was dry. Elution was carried out by adding 100 μl of buffer EB (10 mM Tris, pH 8.5) and the yield was determined photometrically at 260 nm.

Dilution factor 20; Total volume 80 μl. Example 4:

Isolation of plasmid DNA in the presence of LiCI; NaCl or KCI and alcohol in different concentrations 390 μl of each buffer (0.25 - 1.0 M salt; 50 mM Tris, pH 8.5; 15% (w / v) isopropanol) were mixed with 10 μl (4th 10 μg) plasmid DNA (pCMVß; Clontech # 6177-1) (c = 1 μg / μl) mixed and mixed with various amounts of ethanol, which account for up to 71.4% by volume ethanol (see Table 4 below) in the respective binding buffers. After five minutes of incubation at room temperature, the batches were transferred to a column containing a silica membrane and passed through the silica membrane under vacuum (approx. 600 mbar; use of the QIAvac 6S from QIAGEN GmbH). The mixture was then washed with 750 μl of buffer PE (10 mM Tris, pH 7.5; 80% ethanol) and drawn through the membrane until the air was dry. Elution was carried out by adding 100 μl of buffer EB (10 mM Tris, pH 8.5) and the yield was determined photometrically at 260 nm.

Table 4:

Dilution factor 20; Total volume 100 ul Example 5.

Isolation of large amounts of DNA

Increasing amounts of plasmid DNA (c = 1 μg / μl; pCMVb; Clontech # 6177-1) in 15 ml buffer Q1 (1, 25 M NaCl; 50 mM Tris, pH 8.5; 15 vol% Isopropanol) dissolved. Then isopropanol was added to a final concentration of 49.8% by volume, mixed thoroughly and incubated for 5 minutes on the laboratory bench. The DNA / Q1 / isopropanol mixture was transferred to a 20 ml syringe, which was equipped with a syringe filter containing a silica membrane (from Sartorius, Minisart series). The mixture was pressed through the filter under uniform pressure, the filter was removed, and air was repeatedly blown through the membrane with the syringe in order to remove the alcohol residues. For the elution, 5 ml of TE buffer (10 mM Tris, pH 7.5; 1 mM EDTA) were then pressed through the syringe filter using a fresh 5 ml syringe. The yield was determined photometrically at 260 nm. The result is summarized in Table 5.

Table 5:

Dilution factor: 20; Total volume: 5 ml

Result:

The recovery rate of the DNA is between 80 and 90% even with higher amounts of plasmid DNA. Example 6

Isolation of plasmid DNA in the presence of various NaCl concentrations and alcohol

340 μl each of a NaCl solution (0 M; 0.1 M; 0.25 M; 0.5 M; 1 M; 2.5 M; 5 M; saturated solution; 50 mM Tris; pH 8.5) ) with 10μl of a plasmid DNA solution (pCMVß; Clontech # 6177-1) containing 1 μg / μl. 350 μl of isopropanol were added to each sample, which resulted in a concentration of 50% by volume for each batch. After five minutes of incubation at room temperature (20-25 ° C) transferred to a column (QIAvac 6S; QIAGEN GmbH) in which a glass fiber / silica membrane (QIAprep 8-welI strips; QIAGEN GmbH) was arranged. The DNA-containing solutions were passed through the membrane under vacuum (approx. 600 mbar) and then eluted with 200 μl EB buffer (10 mM Tris; pH 8.5). The respective yields of DNA (in percent of the amount used) in the fourfold determinations carried out for each salt concentration and the respective average value are given in Table 6 below.

Table 6

STDEV: standard deviation, based on a sample The result of this experiment suggests that the yield in DNA extraction decreases at very high salt concentrations (5 M, saturated) compared to lower salt concentrations between 0.1 and 2.5 M.

Example 7

Isolation of plasmid DNA in the presence of different salts or different alcohol concentrations

Each 500 μg plasmid DNA was dissolved in 5 ml TE buffer (10mM Tris-Cl; pH 8.0; 1mM EDTA; QIAGEN GmbH) and isopropanol was added until the final concentrations of alcohol in the individual batches at 41.2 , 50, 66.7 and 75 vol%. All determinations for DNA isolation were carried out with a QIAprecipitator Maxi (QIAGEN GmbH) in a QIAvec 6S column (QIAGEN GmbH). Table 7 summarizes the results of the experiments in which the salt concentration in the respective batches was 1.25 M NaCl. The examinations were carried out several times, whereby the results for the individual samples and the mean value calculated therefrom with standard deviation are given in Table 7.

Table 7

Yields [%]

The experiments described above were then modified in such a way that the type of salt solution used was changed, namely the 1.25 M NaCl solution was replaced by a 1.25 M KCI solution. The results of the tests with KCI solution are shown in Table 8.

Table 8

Yields [%]

Finally, the influence of the buffer used on the yield of the plasmid DNA was also examined. The procedure was as described above, and in the batch with a 1.25 M NaCl solution the TE buffer was replaced by a QF buffer (50 mM MOPS; pH 7.0; 15% isopropanol). The results of the multiple determinations together with the mean and standard deviation are summarized in Table 9. The alcohol concentrations in the individual batches which differ from Tables 7 and 8 are due to the 15% isopropanol content of the QF buffer.

Table 9

Yields [%]

The results show that the type of salt used (NaCl / KCI) shows only a minor influence on the yield in DNA isolation. The exchange of the buffer also has practically no influence on the DNA yield (77.4% with 1.25 M NaCl and TE buffer and 75.0% with 1.25 M NaCl and QF buffer).

Claims

claims

1. A method for isolating nucleic acids from a solution, comprising the following steps:

a) adsorbing the nucleic acids contained in the solution in the presence of alkali and / or alkaline earth metal salts on a surface containing Si0 ₂ ,

b) optionally washing the nucleic acids adsorbed on the Si0 ₂ -containing surface with an alcohol-containing washing buffer, and

c) eluting the nucleic acids with an aqueous solution and optionally isolating the nucleic acids,

characterized in that in step a) the adsorption of the nucleic acids on the Si0 -containing surface takes place in the presence of 0.1 to 3 M alkali and / or alkaline earth salts and 37 to 70% by volume of an aliphatic alcohol.

2. The method according to claim 1, characterized in that the nucleic acid is plasmid DNA.

3. The method according to any one of claims 1 or 2, characterized in that the Si0 ₂ -containing surface consists of silica gel, glass fibers or quartz fibers.

4. The method according to any one of claims 1 to 3, characterized in that the SιO ₂ -containing surface is a membrane or a filter.

5. The method according to any one of claims 1-4, characterized in that the alkali salts contained in the solution are halides, preferably NaCl and / or KCI.

6. The method according to any one of claims 1 to 5, characterized in that in step a) NaCl in a concentration of 0.25 to 1.5 M, preferably from 0.5 to 1.25 M and particularly preferably from 0.5 up to 1.0 M is present in the solution.

7. The method according to any one of claims 1 to 6, characterized in that the alcohols contained in the solution are lower aliphatic, branched or unbranched alcohols with a chain length of 1 to 5 carbon atoms.

8. The method according to claim 7, characterized in that the alcohol contained in the solution is ethanol and / or isopropanol.

9. The method according to any one of claims 1 to 8, characterized in that the elution solutions used in step c) morpholinopropanesulfonic acid (MOPS), tris (hydroxymethyl) amino-methane (TRIS) or 2- [4- (2-hydroxyethyl) -1 -piperazino] ethanesulfonic acid (HEPES) in a concentration of 0.001 to 0.5 mol / liter, preferably 0.001 to 0.2 mol / liter, particularly preferably 0.01 to 0.05 mol / liter.

10. Use of a solution containing 0.25-1.5 M NaCl, KCI or a mixture thereof and ethanol or isopropanol in one Concentration of 37 - 70 vol .-% for the adsorption of nucleic acid on a Si0 ₂ -containing surface.

11. Kit for performing the method according to one of claims 1 to 9, containing a) a solution according to claim 10 and b) a Si0 ₂ -containing surface.