DE102017222295B4 - Kits and methods for removing contaminants from a sample containing nucleic acid - Google Patents

Abstract

The present invention relates to a method for the removal of enzyme-inhibiting polyanions, such as humic substances from a sample containing nucleic acid, which is preferably a biological sample or an environmental sample. The invention further relates to a method for isolating nucleic acids from a biological sample or environmental sample with removal of enzyme-inhibiting polyanions. The invention further comprises reagents for carrying out the method according to the invention and a test kit containing these reagents. The invention also relates to the use of an aqueous buffer containing lithium chloride or lithium acetate for the selective elution of nucleic acids from weak anion exchangers with continued binding of the enzyme-inhibiting polyanions.

Description

The present invention relates to a method for the removal of enzyme-inhibiting polyanions, such as humic substances from a sample containing nucleic acid, which is preferably a biological sample or an environmental sample. The invention further relates to a method for isolating nucleic acids from a biological sample or environmental sample with removal of enzyme-inhibiting polyanions. The invention further comprises reagents for carrying out the method according to the invention and a test kit containing these reagents. The invention also relates to the use of an aqueous buffer containing lithium chloride or lithium acetate for the selective elution of nucleic acids from weak anion exchangers with continued binding of the enzyme-inhibiting polyanions.

The microbiological and molecular biological relationships in soils have hardly been researched so far, since most of the microorganisms occurring there are not cultivable in the laboratory. However, biodiversity and metabolic activity can be visualized indirectly via phylogenetic analyzes and transcriptome analytics, with molecular biology methods such as Next Generation Sequencing currently gaining increasing importance. In the case of the necessary isolation of nucleic acids from soil samples and comparable humic substance-containing sample materials, a major problem is the simultaneous purification of humic acids together with the nucleic acid, which sensitively disturb the subsequent analysis of nucleic acids by enzyme-based methods. In particular, the polymerase chain reactions (PCR) but also other enzyme-based methods are inhibited by humic substances. Soil samples are especially relevant as critical samples, for example forensic traces that are contaminated by soil particles.

Humic substances are an inhomogeneous group of compounds that can be divided into different fractions based on their molecular weight. Low molecular weight compounds having a molecular weight of up to 3,000 grams per mole and good solubility in water are referred to as fulvic acids or fulvic acids, high molecular weight compounds of up to several 100,000 grams per mole and decreasing solubility in water, and partial solubility in alkali are referred to as humic acids. Together, the humic substances is a skeleton of aromatic compounds that are connected by bridges. Towards the surface, reactive, predominantly negatively charged, ionic groups predominate, giving polyanionic properties to humic acids. In the combination of these properties: high molecular weight, polyanion, hydrophobic core, the humic acids therefore resemble the nucleic acids and can not be completely separated from nucleic acids by the current purification methods, in particular by anion exchangers.

The prior art describes several ways to separate nucleic acids from humic acids. However, these are usually expensive and require further work steps.

That's how it teaches EP 1 756 136 B1 to separate the humic acids from the nucleic acids a multistage flocculation process. This process is very complicated and involves the risk that nucleic acids are also precipitated by the flocculant. Another approach is through EP 2 499 246 B1 tracked. Here an extraction with organic solvents is described. In addition to the fact that an additional process step is required here, this process has the disadvantage that this extraction does not work for all soils, because the physicochemical properties of nucleic acid and humic acid are too similar.

DE 10 2004 006 115 A1 relates to a process for the preparation of monodisperse acrylic-containing ion exchangers including their use for the separation and purification of biologically active components, such as antibiotics, enzymes, peptides and nucleic acids.

WO 2015/154013 A1 relates to methods and kits for extracting DNA from samples containing humic acid, the isolation preferably being from soil samples.

There is therefore a need for improved methods of separating enzyme-inhibiting polyanions such as humic substances or proteins.

Summary of the invention

• a) Inkontaktbringen der Probe mit einer Anionenaustauscher-Matrix mit schwachen Anionenaustauschergruppen unter Bedingungen die das Anbinden der Nukleinsäure an die Anionenaustauscher-Matrix erlauben,
• b) ein- oder mehrmaliges Waschen der Anionenaustauscher-Matrix mit einem Waschpuffer, wobei die Nukleinsäure an der Anionenaustauscher-Matrix gebunden bleibt, sowie
• c) selektive Elution der an der Anionenaustauscher-Matrix gebundenen Nukleinsäure durch eine Lithiumchlorid oder Lithiumacetat enthaltende Elutionslösung.

Bevorzugte Ausführungsformen der Erfindung ergeben sich aus den abhängigen Ansprüchen.This object is achieved by a method for removing enzyme-inhibiting polyanions from a sample containing nucleic acid, which comprises the following steps:

• a) contacting the sample with an anion exchange matrix having weak anion exchange groups under conditions allowing the binding of the nucleic acid to the anion exchange matrix,
• b) one or more washings of the anion exchange matrix with a wash buffer, wherein the nucleic acid remains bound to the anion exchange matrix, as well as
• c) selective elution of the nucleic acid bound to the anion exchange matrix by means of an elution solution containing lithium chloride or lithium acetate.

Preferred embodiments of the invention will become apparent from the dependent claims.

The process according to the invention combines several decisive advantages over the separation or purification processes known from the prior art.

As the inventors have found, lithium ions in the elution buffer are capable of selectively desorbing the nucleic acids from a weak anion exchange matrix. The enzyme-inhibiting polyanions also bound to the matrix remain bound under these elution conditions and are thus separated from the nucleic acids.

Other alkali metal and alkaline earth metal halides can not effect this separation, which is surprising insofar as, in addition to the pH of the solution, the type and concentration of the anion are particularly relevant in the case of purification by means of a weak anion exchanger. The strong influence of the cation is surprising in this context.

Remarkably, selective elution using lithium salts works for different nucleic acids, both RNA and DNA.

Furthermore, by altering the lithium ion concentration and the pH, the elution solution according to the invention allows a multi-stage elution with increasing elution power to fractionate nucleic acids according to their length. Even in this multistage elution, the interfering polyanions remain bound to the anion exchange matrix, so that highly pure nucleic acid fractions are obtained.

The purification process according to the invention represents a decisive improvement in nucleic acid analysis, since it makes possible a trouble-free analysis of nucleic acids in a simple and uncomplicated manner, and especially for the important field of environmental samples.

The elution solution can be prepared with commercial substances in a simple manner and also inexpensively.

The elution solution can be integrated without additional effort into the already established nucleic acid purification methods.

Thus, the conventional anion exchange matrices can be used and all other process steps established over decades, such as lysis, binding of the nucleic acid or washing of the matrix can be maintained almost unchanged.

Detailed description of the invention

The process of the present invention is based on the ability of aqueous solutions containing lithium cations to selectively desorb and thus elute nucleic acids from a weak anion exchanger, leaving the chemically very similar enzyme-inhibiting polyanions such as humic acids or fulvic acids bound to the anion exchange matrix.

The enzyme-inhibiting polyanions separable by the process of the present invention are preferably selected from the group consisting of a polyphenol, a humic substance, a humic acid, a fulvic acid, a humic acid and a vegetable phenol, or mixtures thereof.

Particularly in the case of soil samples, complex polyanion mixtures are present, which in addition to humin also comprise humic acid and fulvic acid.

In a preferred embodiment, the method of the invention is used on a sample which is or derived from an environmental or biological sample.

Most preferably, this sample was taken from food, soil, sediment, rocks, rocks, mud, compost, rotting biological substances, a forensic specimen, archaeological remains, peat, compost, oil, water, terrestrial or underground water, atmospheric and industrial Water, dust, commercial pottery mixes, or soil changes or deep-sea potholes.

According to the invention, the anion exchanger is a matrix which carries weak anion exchange groups. These are preferably primary, secondary or tertiary amines.

The weak anion exchange groups are preferably selected from the group comprising aminomethyl (AM), aminoethyl (AE), methylhydroxyaminoethyl (MAE), aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, diethylaminoethyl (DEAE), ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, trimethylamino (TMA) , Triethylaminoethyl (TEAE), linear or branched polyethylenimine (PEI), carboxylated or hydroxyalkylated polyethylenimine, Jeffamine, Tris, bis-tris, spermine, spermidine, 3- (propylamino) propylamine, polyamidoamine (PAMAM) dendrimers, polyallylamine, polyvinylamine, N- Morpholinoethyl, polylysine, tetraazacycloalkanes, cyclic amines and protonatable aromatic amines.

In a particularly preferred manner DEAE and in particular MAE is used here.

The anion exchange material may be packed in a column or freely in solution. Preference is given to the execution as a packed material in a column. Preferred formats for the material are particles, beads, membranes, filters, plates and capillary tubes.

The anion exchange material may conveniently be provided with one or more prefilters. As the filter material, for example, and not exclusively, cellulose or sintered polyethylene may be used.

The anion exchange group is bound to a matrix as a solid surface in the method of the invention. The connection between the solid phase and the anion exchange group can be effected by a so-called spacer (so-called spacer) of variable length. Such a spacer, which may be linear or branched, forces a spatial distance between the matrix and the anion exchange group and thus overcomes steric disturbances of the ligand-ligate interaction. The spacer can comprise 3 to 30 carbon atoms.

As the matrix, any material suitable for anion exchange chromatography can be used, such as silica, boron silicates, organic polymers, polysaccharides, metal oxides, metals, Sephadex, Sepharose, hydrogels such as agarose.

The matrix as solid phase is preferably silica gel. The silica gel may have an average particle size of less than 200 μm, preferably less than 100 μm and more preferably less than 60 μm.

The anion exchange material and the filter elements which may be used are preferably equilibrated in the process according to the invention with a solution which consists of water and a surface-active substance, for example a detergent or a C 1 -C 4 alcohol, and to slightly acidic pH values, preferably from pH 5.0 to pH 6.9 is set.

lysis step

The nucleic acid-containing sample used in the method is usually obtained by a cell disruption, the so-called "lysis".

Conveniently, the DNA-containing cells contained in the sample, which is preferably a soil sample or soil contaminated sample, are lysed in a lysis buffer by a method known to those skilled in the art, for example by bead beating, mortars under liquid nitrogen, or heating. to release the nucleic acid contained in the cells. This is done by the lysis buffer the optionally contained in these samples additionally free nucleic acid also brought into solution. Inevitably, in addition, with appropriately loaded samples, the fraction of fulvin and humic acids is brought into solution, which then contaminate the nucleic acid-containing solution.

Suitable lysis buffers are known to the person skilled in the art. The lysis buffer in this case contains at least one detergent, which is preferably an anionic detergent. Particularly preferred here is the use of sodium dodecyl sulfate (SDS).

The detergent, which is preferably SDS, in one embodiment is present at a concentration of less than 10% (m / v), preferably less than 5% (m / v), more preferably less than 3% (m / v).

Furthermore, the lysis buffer may contain a complexing agent which is, for example, but not limited to, ethylenediaminetetraacetate (EDTA) or citrate.

The chelator, which is preferably citrate or EDTA, is contained in the lysis buffer at a concentration of less than 500 millimoles per liter (mM), preferably less than 200 mM, more preferably less than 150 mM.

As further ingredients of the lysis buffer, buffer substances, for example Tris, MOPS, acetate, citrate, bis-tris or MES, can be present in sufficient concentration to stabilize the pH.

The concentration of the buffer substance, in which case Tris is preferred, is for example less than 500 mM, preferably less than 200 mM and particularly preferably less than 150 mM.

The pH of the lysis buffer is in the range of pH 5.0 to 9.0, preferably in the range of pH 6.0 to 8.0 and more preferably pH 7.0.

The lysis buffer can be supplemented in the process according to the invention by an organic solvent, for example phenol: chloroform: isoamyl alcohol (25: 24: 1 v / v / v).

In one embodiment, the beads used for cell disruption by bead-beating have a diameter of less than 3.0 mm, preferably less than 2.0 mm, more preferably less than 1.0 mm.

The beads can be made of one of the following materials: steel, glass, ceramic or corundum. Preference is given here beads made of ceramic, and particularly preferably zirconium with an yttrium content between 0 and 10%.

The cell digestion is particularly preferably carried out in the presence of the sample, the lysis buffer, optionally the organic solvent and the beads by shaking, for example and not exclusively horizontally at 30 Hz for 5 min, vertically at 5 m / s for 30 s or orbital at 2700 revolutions per minute with a radius of 3 mm for 5 min.

Optimal conditions are sample-specific and easy for the skilled person to determine.

Following cell disruption, undissolved soil particles are separated from the dissolved components, for example by centrifugation or by filtration. The nucleic acid-containing clear supernatant can be mixed in the process according to the invention with a further solution, with which the binding conditions are defined to the anion exchange matrix, if this is not already done by the composition of the lysis buffer. This is a salt solution, preferably a solution containing carboxylic acid salt, more preferably a potassium acetate solution.

Volume and concentration are expediently chosen so that in the resulting solution of lysis supernatant and binding solution, a potassium and acetate concentration of more than 100 mM, preferably more than 200 mM, more preferably more than 300 mM and preferably less than 750 mM, particularly preferred less than 500mM.

The resulting pH is suitably at pH 4.0 to pH 7.0, preferably at pH 4.5 to 6.5, more preferably at pH 5.0 to pH 6.0.

connection step

The lysis supernatant, in which the appropriate binding conditions have been set, is contacted with the (preferably equilibrated) anion exchange material. In the embodiment as packed material in a column, the solution is contacted via an entrance to the anion exchange material and leaves the packing via a downstream exit.

Driving force may be gravitational flow or positive pressure mediated by, for example, a pump. Negative pressure (vacuum) is preferably not used. Particularly preferred is the embodiment as a gravity flow column.

If the anion exchange material is in bulk, it will be contacted with the lysis supernatant, for example by suspending the anion exchange matrix in the lysis supernatant. After binding, the anion exchange material is removed from the lysis supernatant, for example, by centrifugation, filtration or, if possible due to the choice of the solid phase, a magnetic field. In binding, polyanions such as nucleic acids, humic acids and negatively charged proteins are at least partially bound to the positively charged anion exchange matrix.

If necessary, a prefilter used is rinsed out to also bind the nucleic acid contained therein in the dead volume to the anion exchange matrix. The rinsing solution typically used is a solution whose composition corresponds to the solution with which the nucleic acid was bound to the anion exchanger. Alternatively, water can be used without further additives.

wash

By means of a washing step, first contaminants, for example proteins or fulvic acids, are eluted from the anion exchanger in the process according to the invention.

In this case, a washing solution is used, which promotes the composition of the elution of low molecular weight oligo- or polyanions, however, high molecular weight polyanions keeps bound to the column. In the case of an anion exchanger, the pH value is of crucial importance. The usable concentration range of the salt used for the washing step becomes narrower, the higher the pH of the solution, since the number of positively charged groups of the anion exchanger changes depending on the pKa value of the anion exchange group and the pH of the solution. At alkaline pH, the proportion of protonated, ie positively charged anion exchange groups decreases, which promotes elution of bound anions.

Since MAE is particularly preferably used as the anion exchange group in the process according to the invention, the wash solution used preferably has a pH of from 6.5 to 7.5, particularly preferably of pH 7.0, the pH being limited by a buffer substance, for example Tris -Cl is buffered. In this case, low molecular weight contaminants are eluted by a saline solution having an anion concentration of from 200 mM to 600 mM, preferably from 300 mM to 500 mM, more preferably from 450 mM. As the salt, for example, halides, phosphates, sulfates, cyanates and acetates can be used. Guanidinium thiocyanate is particularly preferably used. To better impart interfacial activity and to adjust the optimum polarity of the liquid phase, the wash solution contains ethanol in a concentration of 5 to 25% by volume, preferably 10 to 20% by volume, and more preferably 15% by volume.

elution

In the process of the invention, the elution of the nucleic acid from the anion exchanger is carried out by an elution solution containing lithium chloride (LiCl) or lithium acetate (LiOAc) as a salt.

In a preferred embodiment, the elution solution contains the lithium chloride (LiCl) or lithium acetate as sole salt. This lithium salt is optionally supplemented by an ion in low concentration, which acts as a buffer substance for the pH and / or a C1-C4 alcohol to adjust the polarity.

If desired, elution can be carried out in several stages with increasing elution force to fractionate nucleic acids according to their length.

If total nucleic acid is to be isolated according to the method according to the invention, the elution pH value is dependent on 700 mM to 1500 mM LiCl at a pH of 7.0 to 10.0, preferably at pH 7.5 to 9 when using lithium chloride , 0, more preferably at pH 8.0 and 850 mM LiCl. The buffer substance used here is preferably Tris-Cl in a concentration of, for example, 100 mM.

In the fractionation process according to the invention, the elution of short DNA to about 3000 base pairs (bp) or of RNA to about 3000 nucleotides (nt) at pH 7.0 and a salt concentration of 1000 mM, longer nucleic acid is carried out with 850 mM LiCl at pH 8.0 eluted. The pH is particularly preferably stabilized by Tris-Cl in a concentration of 100 mM. As the pH increases, elution of longer nucleic acids occurs at the same LiCl concentration, so that the LiCl concentration must be lowered at a higher pH if fractionation is required. The skilled person can determine appropriate conditions by simple experiments. For better imparting interfacial activity and adjusting the solvent polarity, the elution solution optionally contains a C1-C4 alcohol, preferably ethanol or isopropanol, in a concentration of 1 to 25% by volume, preferably 10 to 20% by volume, and especially preferably 15% by volume.

post-treatment step

The desalting and concentration of the eluted nucleic acid is carried out, for example, by a method known to the person skilled in the art, for example by precipitation with isopropanol. For this purpose, the Anionenaustauschereluat is mixed with 0.7 volume of isopropanol, incubated for 0 to 24 hours at room temperature and then preferably filtered on a glass or quartz fiber filter ("silica membrane"). Corresponding devices are known to the person skilled in the art.

The glass fiber filter used may be, for example and not exclusively, a binder-free glass fiber filter having a weight of 120 g / cm ² , 0.6 mm thickness and an average particle retention capacity in solutions of 1.4 μm, one or more of which several layers are used for filtration. The filtration can be accelerated by vacuum, centrifugation or by positive pressure.

The retained on the filter nucleic acid precipitate is washed by one or more washing steps with ethanol in a concentration of preferably more than 69% in water. Remaining ethanol is removed by centrifugation or air drying. The nucleic acid precipitate is then dissolved in water, TE buffer or any suitable solvent and eluted from the filter.

Alternatively, the precipitation can take place without a glass fiber filter directly in a reaction vessel known to the person skilled in the art.

1. a) eine Anionenaustausch-Matrix mit schwachen Anionenaustauschergruppen;
2. b) einen Waschpuffer;
3. c) einen Lithiumchlorid oder Lithiumacetat enthaltenden Elutionspuffer; und
4. d) Anweisungen zur Durchführung des erfindungsgemäßen Verfahrens.

In a second aspect, the invention provides a kit for isolating nucleic acids according to the method of the invention, the kit comprising:

1. a) an anion exchange matrix with weak anion exchange groups;
2. b) a wash buffer;
3. c) an elution buffer containing lithium chloride or lithium acetate; and
4. d) instructions for carrying out the method according to the invention.

1. 1. einen Lysepuffer
2. 2. Isopropanol
3. 3. Glasfilter oder Silikamembran-Filter
4. 4. Röhrengefäße.

The kit for isolating non-nucleic acids according to the method of the invention may also comprise the following optional components:

1. 1. a lysis buffer
2. 2. isopropanol
3. 3. Glass filter or silica membrane filter
4. 4. Tubular vessels.

In a third aspect, the invention relates to the use of a lithium chloride-containing elution buffer for removing enzyme-inhibiting polyanions from a nucleic acid-containing sample by selective elution of nucleic acids bound to an anion exchange matrix having weak anion exchange groups with remaining attachment of the enzyme-inhibiting polyanions to the anion exchange matrix ,

definitions

In the context of the present invention, an "enzyme-inhibiting polyanion" is understood as meaning a polymer which carries a plurality of anionic groups at a pH of 7.0 and which exerts a negative influence on the amplification of the nucleic acid in a PCR.

According to the invention, a "humic acid" is a high molecular chemical compound which, among other humic substances, are formed by "humification" during the degradation process of biological material. The molar mass range of humic acid is between 2000 and 300,000 daltons. It consists mainly of partially degraded vegetable lignin and cellulose, which are often attached to proteins and carbohydrates. The humic acid typically contains different chemical components, such as quinone, phenol, sugar or peptide components, which are preferably linked together via phenolic bridges.

In the context of the invention, a weak anion exchange group is defined as an anion exchanger that is ionizable only in a limited pH range. These are preferably primary, secondary or tertiary amines.

embodiments

Isolation of DNA and RNA from a soil sample

The following example describes by way of example an inventive purification process using a soil sample and shows the advantageous purification compared to standard elution buffers.

Test procedure

• Schritt 1 (Fraktion 1)
  1. 1. 1000 mM LiCI, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  2. 2. 1000 mM NaCl, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  3. 3. 1000 mM KCl, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  4. 4. 1000 mM NH₄Cl, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  5. 5. 1000 mM Guanidinium-Cl (GuCI), 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  6. 6. 500 mM MgCl₂, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
  7. 7. 500 mM CaCl₂, 100 mM Tris-Cl, pH 7,0, 15% Ethanol
• Schritt 2 (Fraktion 2)
  1. 1. 850 mM LiCI, 100 mM Tris-Cl, pH 8,0
  2. 2. 850 mM NaCl, 100 mM Tris-Cl, pH 8,0
  3. 3. 850 mM KCl, 100 mM Tris-Cl, pH 8,0
  4. 4. 850 mM NH₄Cl, 100 mM Tris-Cl, pH 8,0
  5. 5. 850 mM Guanidinium-Cl (GuCI), 100 mM Tris-Cl, pH 8,0
  6. 6. 425 mM MgCl_e, 100 mM Tris-Cl, pH 8,0
  7. 7. 425 mM CaCl₂, 100 mM Tris-Cl, pH 8,0

Per batch, 500 mg of soil sample were placed in a vessel containing 1.5 g of ceramic beads. After addition of 800 μL lysis buffer and 100 μL phenol: chloroform: isoamyl alcohol (25: 24: 1 v / v / v), the cells contained in the sample were disrupted by bead-beating. The samples were separated by centrifugation into a nucleic acid-containing supernatant and a pellet of soil particles and beads. The supernatants were transferred to new reaction vessels and mixed with a binding buffer. After another centrifugation step for fine clarification, the supernatants were combined in a common vessel and mixed by vortexing to give a homogeneous master lysate. Lysis-related differences in nucleic acid concentration and humic acid contamination are leveled by this step. From this master lysate with uniform nucleic acid and humic acid concentrations, equal volumes were "gravity flowed" onto seven identical, equilibrated MAE anion exchange columns. Low molecular weight contaminants were removed by adding a wash buffer. The elution of the seven columns was done in two steps with the following solutions:

• Step 1 (Fraction 1)
  1. 1. 1000 mM LiCl, 100 mM Tris-Cl, pH 7.0, 15% ethanol
  2. 2. 1000 mM NaCl, 100 mM Tris-Cl, pH 7.0, 15% ethanol
  3. 3. 1000 mM KCl, 100 mM Tris-Cl, pH 7.0, 15% ethanol
  4. 4. 1000 mM NH ₄ Cl, 100 mM Tris-Cl, pH 7.0, 15% ethanol
  5. 5. 1000 mM guanidinium Cl (GuCl), 100 mM Tris-Cl, pH 7.0, 15% ethanol
  6. 6. 500 mM MgCl ₂ , 100 mM Tris-Cl, pH 7.0, 15% ethanol
  7. 7. 500 mM CaCl ₂ , 100 mM Tris-Cl, pH 7.0, 15% ethanol
• Step 2 (Fraction 2)
  1. 1. 850 mM LiCl, 100 mM Tris-Cl, pH 8.0
  2. 2. 850mM NaCl, 100mM Tris-Cl, pH 8.0
  3. 3. 850mM KCl, 100mM Tris-Cl, pH 8.0
  4. 4. 850 mM NH ₄ Cl, 100 mM Tris-Cl, pH 8.0
  5. 5. 850 mM guanidinium Cl (GuCl), 100 mM Tris-Cl, pH 8.0
  6. 6. 425 mM MgCl _e , 100 mM Tris-Cl, pH 8.0
  7. 7. 425 mM CaCl ₂ , 100 mM Tris-Cl, pH 8.0

The eluates are collected and desalted by means of known isopropanol precipitation and concentrated. For this purpose, 0.7 volumes of isopropanol are added to the eluates. By filtration on a glass fiber filter, the nucleic acid precipitate is collected and washed with a solution of 80% ethanol in water. After the ethanol removal by drying, the nucleic acid is dissolved in 100 μL of water (H ₂ O _bidest. ) And used for the following experiments.

Analysis of the eluates

Photometric measurement

A photometric measurement of the total eluates at 300 nm indicates the degree of browning of the eluates. The higher the absorbance at 300 nm, the brighter the eluates, indicating the carryover of humic acids. Preference is therefore as small as possible values below 0.05.

The results are shown in Table 1 and shown. These show that clean, photometrically uncolored eluate fractions with an absorbance at 300 nm of less than 0.05 are obtained only when using LiCl. The use of sodium chloride-containing standard elution buffer leads to eluates with 3 to 4 times higher humic acid concentration. Table 1: Extinction of the eluate fractions at 300 nm LiCl NaCl KCl NH ₄ Cl GuCl MgCl ₂ CaCl ₂ Fraction 1 0,044 0.174 0,248 0.104 0,298 0.671 0.362 Fraction 2 0,042 0.131 0.221 0,060 0,155 0.123 0.154 (Table 1 and ).

Elelectrophoretic analysis

20 μL of the first elution fraction and 10 μL of the second elution fraction are separated on a 1% TAE agarose gel. The gel image is in shown. The difference in nucleic acid elution is evident, although in all cases the same concentration of chloride ions has been present. RNA can be recognized by the ribosomal bands of 16S and 23S rRNA, DNA is visible as a high molecular weight cloud and low molecular weight "smear". As a marker of GeneRuler 1 kb marker is plotted ^® (Fa. Thermo Fisher Scientific GmbH, Dreieich, FRG).

With NH4 as cation, no significant nucleic acid elution can be detected. Magnesium and calcium mainly elute DNA, less RNA. Potassium and guanidinium elute delays RNA and no DNA.

Remarkably, fractionated purification of RNA (1st fraction) and DNA (2nd fraction) is possible only with the lithium-containing buffer.

Analysis of enzyme inhibition by RT-PCT

To demonstrate enzymatic inhibition, an RT-PCR is first carried out. It is known that humic substances can inhibit thermostable DNA polymerases such as TAQ polymerase.

RT-Primer: MYT4- 797R PCR16
5'-CAGCTTGGTAGAATCGATCAGCTAC/GGACTACCAGGGTATCTAATCCTGTT-3'

PCR-Primer/ Forward Primer: 331F PCR16
 5'-TCCTACGGGAGGCAGCAG-3'

PCR-Primer/ Reverse Primer: MTY4 PCR Uni:
5'-CAGCTTGGTAGAATCGATCAGCTAC-3'

In the present approach, a 466 bp piece of 16S rRNA is amplified. By selecting the primers "331F PCR16" and "MTY4 PCR Uni" only RNA is amplified. For the transcription of the RNA into the cDNA by means of the enzyme reverse transcriptase, the primer "MYT4-797R PCR16" is used.

 RT primer: MYT4-797R PCR16
5'-CAGCTTGGTAGAATCGATCAGCTAC / GGACTACCAGGGTATCTAATCCTGTT-3 ' 

 PCR Primer / Forward Primer: 331F PCR16
 5'-TCCTACGGGAGGCAGCAG-3 ' 

 PCR primer / reverse primer: MTY4 PCR Uni:
5'-CAGCTTGGTAGAATCGATCAGCTAC-3 ' 

The amplification and quantification of the amplificates is carried out by means of real-time PCR using the "BioLine SensiFAST SYBR low-Rox One Step" kit (Bioline). The volume of the RT-PCT preparations is 20 μL.

The results of the RT-PCR for the first eluate fraction are in shown. The eluate fraction 1 shows a clear signal only when using LiCl and in the positive control (POS). When using NaCl for elution, despite gelelektrophoretisch comparable yield of 16S rRNA (s. ) to detect a drastically delayed signal with non-exponential amplification, which is a sign of reaction inhibition.

In a PCR, the number of DNA theoretically doubles on each cycle (2 ⁿ with n = number of cycles), so a difference of 12.6 cycles (20.91-33.51) until the Cp value is reached mathematically a concentration difference of factor 212.6 = about 6000 would correspond. The Cp values of in shown RT-PCR for the 1 Elulatfraktion and in shown RT-PCR for the 2 Elulatfraktion that are shown in the following table: Table 2: Cp values of the eluate fractions in the 16S rRNA - RT-PCR LiCl NaCl KCl NH ₄ Cl GuCl MgCl ₂ CaCl ₂ Fraction 1 12.67 20.91 - 33.51 - - - Fraction 2 14.69 25.24 - 28,11 > 36 23.64 21.51

Such a difference in concentration can not be detected in the gel image, where the 16S rRNA bands of both eluates have the same intensity. Consequently, the big difference in the PCR can only be explained by the fact that the theoretically possible duplication did not take place every cycle, but the activity of the polymerase in the NaCl eluate fraction was reduced (x ⁿ with x << 2).

Analysis of the second eluate fraction showed only a measurable 16S rRNA amplicon for the eluates of lithium, sodium, potassium, guanidinium, and magnesium (see ). The corresponding RT-PCR in again shows only an exponential increase in lithium. When using the other salts, the signals are much delayed and with a more linear increase, a clear indicator of inhibition.

pA: 5'-AGAGTTTGATCCTGGCTCAG-3'

pH': 5'-AAGGAGGTGATCCAGCCGCA-3'

To detect a PCR inhibition in the DNA fraction, the 16S rRNA gene is completely amplified at about 1.6 kbp. The primer used for this is the known primer pair pA and pH '.

 pA: 5'-AGAGTTTGATCCTGGCTCAG-3 ' 

pH ': 5'-AAGGAGGTGATCCAGCCGCA-3' 

PCR is performed by endpoint PCR and 35 cycles. The eluates are used undiluted and diluted 1:10 with water in the PCR. A PCR inhibition is shown in that the PCR shows an amplificate only at higher dilution levels.

Undiluted, a signal is measurable only when lithium is used ( ). According to However, especially when using MgCl ₂ and CaCl _2, a high DNA yield and thus an intensive PCR amplificate would have been expected. The conspicuous high E300 (see Table 1) and the intensive brown coloration of the eluates indicated by this indicate consequently a strong metabolism of humic substances and as a consequence an inhibition of the PCR.

In a 1: 100 dilution ( ) are evident in other samples significant bands. By dilution, therefore, the amplifiability in a PCR could be restored, indicating a PCR inhibition of the undiluted samples.

On the basis of photometric determination of the brown color of the eluates (humic acid carryover) and PCR data, it is thus possible to detect a carry-over of humic acid from the soil sample into the eluates. This occurred in all salts used for elution, but not in lithium chloride. The chloride concentration and thus the relevant component for an anion exchanger was uniform in all samples. The clear influence of the cation is unexpected and shows a strong positive effect.

list of figures

• 1 shows the results of the isolation of RNA and DNA from a humic acid-containing soil sample according to Example 1 with analysis of the extinction of the RNA eluates (fraction 1) and DNA eluates (fraction 2) at 300 nm.
• 2 shows the results of the isolation of RNA and DNA from a humic acid-containing soil sample according to Example 1 by means of a gel electrophoretic analysis of the eluates (fraction 1 and 2). Shown is the ethidium bromide stained agarose gel under UV exposure. The size marker was a 1kb ladder.
• 3 shows the results of the isolation of RNA from a humic acid-containing soil sample according to Example 1 by means of a semiquantitative RT-PCR analysis of the eluates. Shown are the amplification curves of the individual PCR reactions.
• 4 shows the results of the isolation of DNA from a humic acid-containing soil sample according to Example 1 by means of a semiquantitative RT-PCR analysis of the eluates. Shown are the amplification curves of the individual PCR reactions.
• 5 Figure 3 shows the results of isolating DNA from a humic acid-containing soil sample according to Example 1 by end-point PCR analysis (35 cycles) of the undiluted eluates. Shown is an ethidium bromide stained agarose gel of the separated PCR reaction mixtures. The size marker was the "GeneRuler 1kb DNA ladder" (size in bp: 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 8000, 10.000).
• 6 Figure 3 shows the results of isolation of DNA from a humic acid-containing soil sample according to Example 1 by end-point PCR analysis (35 cycles) of 1:10 diluted eluates. Shown is an ethidium bromide stained agarose gel of the separated PCR reaction mixtures. The size marker was the "GeneRuler 1kb DNA ladder" (size in bp: 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 8000, 10.000).
• 7 Figure 2 shows the results of isolation of DNA from a humic acid-containing soil sample according to Example 1 by end-point PCR analysis (35 cycles) of the 1: 100 diluted eluates. Shown is an ethidium bromide stained agarose gel of the separated PCR reaction mixtures. The size marker was the "GeneRuler 1kb DNA ladder" (size in bp: 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 8000, 10.000).

Other variants of the invention and their execution will become apparent to those skilled in the art from the foregoing disclosure, the drawings and the claims.

Terms used in the claims, such as "comprising," "comprising," "including," "containing," and the like, do not exclude other elements or steps. The use of the indefinite article does not exclude a majority. A single device can perform the functions of several units or devices mentioned in the claims. Reference signs indicated in the claims should not be regarded as limitations on the means and steps employed.

Claims (11)

A method of removing enzyme-inhibiting polyanions from a sample containing nucleic acid comprising the following steps: i. Contacting the sample with an anion exchange matrix having weak anion exchange groups under conditions allowing the binding of the nucleic acid to the anion exchange matrix, ii. washing the anion exchange matrix one or more times with a washing buffer, the nucleic acid remaining bound to the anion exchange matrix, and iii. selective elution of the bound to the anion exchange matrix nucleic acid by a lithium chloride or lithium acetate-containing elution solution. Method according to Claim 1 , characterized in that the enzyme-inhibiting polyanions are selected from the group consisting of a polyphenol, a humic substance, a humic acid, a fulvic acid, a humic acid and a vegetable phenol, or mixtures thereof. Method according to Claim 1 or 2 characterized in that the sample comprises an environmental or biological sample preferably extracted from a foodstuff, soil, sediment, rocks, rocks, mud, compost, rotting biological substances, a forensic specimen, archaeological remains, peat, compost, Oil, water, terrestrial or subterranean water, atmospheric and industrial water, dust, commercial pottery mixtures, or soil alterations or deep-sea sediments. Method according to Claim 1 to 3 characterized in that the weak anion exchange groups are selected from the group comprising aminomethyl (AM), aminoethyl (AE), methylhydroxyaminoethyl (MAE), aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, diethylaminoethyl (DEAE), ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, Trimethylamino (TMA), triethylaminoethyl (TEAE), linear or branched polyethyleneimine (PEI), carboxylated or hydroxyalkylated polyethylenimine, Jeffamine, Tris, Bis-Tris, spermine, spermidine, 3- (propylamino) propylamine, polyamidoamine (PAMAM) dendrimers, polyallylamine, Polyvinylamine, N-morpholinoethyl, polylysine, tetraazacycloalkanes, cyclic amines and protonatable aromatic amines, with DEAE being preferred and MAE being particularly preferred. Method according to one of the preceding claims, characterized in that the matrix having the weak anion exchange groups has one or more of the following properties: a) The weak anion exchange groups are attached via a spacer to a matrix, which preferably has a length of 3 to 30 carbon atoms having; b) The matrix is silica gel with an average particle size of less than 200 μm, preferably less than 100 μm and particularly preferably less than 60 μm; c) the matrix with the weak anion exchange groups is equilibrated before contacting in step i) with a solution which consists of water, a surfactant and / or a C 1 -C 4 alcohol and preferably has a pH of between 4.0 and 6, 9 has. A method according to any one of the preceding claims, characterized in that the washing buffer is a buffered saline solution having one or more of the following characteristics: a) a buffer solution having a pH of 6.5 to 7.5, and preferably of 7.0 ; b) an anion concentration of from 200 mM to 600 mM, preferably from 300 to 500 mM and more preferably from 450 mM; c) as salt anions, halides, phosphates, sulfates, cyanates, acetates and thiocyanates; d) as a salt guanidinium thiocyanate; e) an alcohol, which is preferably ethanol in a concentration of 5 to 25 vol .-%, preferably from 10 to 20 vol .-% and particularly preferably from 15 vol .-%. A method according to any one of the preceding claims, characterized in that the elution buffer is a buffered lithium chloride or lithium acetate solution having one of the following properties: a) upon elution of total nucleic acid lithium chloride or lithium acetate at a concentration of 700-1500 mM at a pH Value from 7.0 to 10.0, preferably at pH 7.5 to 9.0, and more preferably at pH 8.0 and 850 mM lithium chloride; b) upon elution of short double-stranded DNA to about 3000 base pairs of lithium chloride at a concentration of 1000 mM at a pH of 7.0; c) upon elution of RNA to about 3000 nucleotides of lithium chloride in a concentration of 1000 mM at a pH of 7.0; d) upon elution of long double-stranded DNA greater than 3000 base pairs, lithium chloride at a concentration of 850 mM at pH 8.0; e) when RNA of greater than 3000 nucleotides is eluted, lithium chloride in a concentration of 850 mM at a pH of 8.0, wherein tris (hydroxymethyl) -aminomethane (Tris) is preferably used as a buffer substance, and more preferably at a concentration of 100 mM; and wherein the elution buffer optionally has a C1-C4 alcohol, which is preferably ethanol or isopropanol, in a concentration of 1 to 25% by volume, preferably of 10 to 20% by volume and particularly preferably of 15% by volume , Method according to one of the preceding claims, characterized in that the method prior to contacting in step i) comprises cell disruption by treating the nucleic acid-containing cells contained in the starting material with a lysis buffer, the cell digestion having one or more of the following properties: a) the lysis buffer contains at least one detergent which is preferably an anionic detergent and more preferably sodium dodecyl sulfate (SDS) b) the lysis buffer contains SDS at a concentration of less than 10% (m / v), preferably less than 5% (m / v) and more preferably less than 3% (m / v); c) the lysis buffer contains a chelator at a concentration of less than 500 mM, preferably less than 200 mM, and more preferably less than 150 mM, with citrate or EDTA being the preferred chelator; d) the lysis buffer contains a buffer substance which is preferably selected from the group comprising Tris, MOPS, acetate, citrate, bis-Tris and MES; e) the lysis buffer contains the buffer substance in a concentration of less than 500 mM, preferably less than 200 mM and more preferably less than 150 mM, f) the pH of the lysis buffer is in the range of pH 5.0 to 9 , 0, preferably between 6.0 and 8.0, and particularly preferably at pH 7.0. g) the lysis buffer may be added to a mixture of phenol: chloroform: isoamyl alcohol (25: 24: 1 v / v / v); h) the cells can be disrupted by bead-beating, mortars under liquid nitrogen or by heating. Process according to any one of the preceding claims, characterized in that it further comprises after elution in step iii): d) demineralization and concentration of the eluted nucleic acid by precipitation with isopropanol, filtration through a glass or quartz fiber filter ("silica membrane") and optional washing steps followed by drying and elution from the filter and / or analysis of the nucleic acid. Kit for isolating nucleic acids according to any one of Claims 1 to 9 the kit comprising: a) an anion exchange matrix having weak anion exchange groups; b) a wash buffer; c) an elution buffer containing lithium chloride or lithium acetate; d) instructions for carrying out the method according to one of the Claims 1 to 17. Use of a lithium chloride-containing elution buffer for the removal of enzyme-inhibiting polyanions from a nucleic acid-containing sample by selective elution of bound to an anion exchange matrix with weak anion exchange groups nucleic acids with remaining binding of enzyme-inhibiting polyanions on the anion exchange matrix.

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