Patent Application
20130023655
The isolation of high-quality nucleic acids is a prerequisite for many different techniques in modern molecular biology, such as PCR amplification, blotting analysis and genomic-library construction, used for example in the field of molecular diagnostics. Especially if the nucleic acids are obtained from biological samples containing cellular material, it is necessary to separate them from contaminants like proteins, lipids and other cellular constituents that otherwise may interfere with restriction enzymes, ligases, and/or thermostable DNA polymerases used in these downstream applications. Furthermore, RNA nucleases (RNases) and particularly DNA nucleases (DNases) present in biological samples have to be removed to prevent degradation of the DNA.
A variety of different methods have been developed for the isolation of genomic DNA from biological samples containing cellular components. All of these methods involve a step of disrupting and lysing the starting material by breaking the cellular membrane and releasing its contents into solution. The mixture obtained is called lysate. In the following steps proteins, in particular nucleases, and the contaminants of the mixture and/or the used solutions are removed from the lysate, and finally the (more or less) purified DNA has to be recovered (an overview can be found in the QIAGEN brochure on “Genomic DNA Purification”). The step of purifying the DNA is of utmost importance, as carryover of mixture or buffer contaminants such as salts, detergents, organic solvents, in particular phenol and ethanol, often inhibit performance of DNA in downstream applications.
Anionic surfactants are known for having excellent protein denaturing and solubilizing properties. Particularly Sodium dodecyl sulfate (SDS) is an anionic surfactant which is used as an active agent in various lysis buffers. Its almost complete removal from a sample on the other hand is quite challenging, but necessary, as even small residual amounts of SDS severely interfere in down-stream applications such as PCR amplification and chromatographic methods such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), and the like.
Nevertheless, many nucleic acids containing samples obtained from cell lysates and purified using procedures known in the state of the art either still contain considerable amounts of dodecyl sulfate ions (DS−) or contaminants such as salts or organic solvents used to separate the nucleic acids from the dodecyl sulfate ions in addition to further buffer or cell components.
A very simple and fast technique for the isolation of genomic DNA from cell lysates is to incubate the cell lysates at high temperatures, e.g. at 90° C. for about 20 min or to directly use the lysates after an additional protease digestion. While nuclease enzymes are inactivated using this process, the lysates obtained still comprise buffer components such as dodecyl sulfate ions and salts, acting as enzyme inhibitors in downstream application such as PCR and the like. Accordingly these quick and dirty techniques are only appropriate for a limited range of applications.
So-called salting-out methods, wherein proteins and other contaminants are precipitated from the crude cell lysate by adding a solution comprising a high concentration of a salt, such as potassium acetate or ammonium acetate, are well-known techniques for separating DNA from other cellular components present in a cell lysate. The precipitates formed are then removed from the solution comprising the DNA by centrifugation, and the DNA is recovered from the supernatant by precipitation with alcohol in a further step. In these methods, removal of proteins, in particular nucleases, and other contaminants often is quite inefficient, and an additional RNase treatment, a dialysis and/or repeated precipitations by alcohol are necessary to obtain DNA sufficiently pure to be used in downstream applications, which renders the methods tedious and time-consuming.
Another possibility to separate nucleic acids from the other compounds present in a cell lysate is to extract the contaminants using organic solvents. In a first step, the cells typically are lysed using a detergent, and the lysates are then extracted using solvents, such as phenol, chloroform, and isoamyl alcohol, to remove the contaminants. The toxicity of the solvents used is one drawback of these methods. Furthermore, special attention has to be paid to the pH and salt concentration to ensure that the majority of contaminants are extracted into the organic phase, while the nucleic acids remain within the aqueous phase. The nucleic acids are then recovered from the aqueous phase by alcohol precipitation. Even though organic extraction methods are very time-consuming, the nucleic acids isolated using these methods often contain residual phenol and/or chloroform, which act as inhibitors in downstream applications such as PCR. In addition, toxic waste is generated which has to be disposed in accordance with hazardous waste guidelines.
In recent years sorption procedures based on ion exchange, affinity and/or hydrophobic interactions have been developed in order to minimize DNA degradation during purification. In these sorption procedures, the DNA is more or less specifically “sorbed”, that is either adsorbed, absorbed or chemically bound, to a stationary solid phase, comprising a resin or a matrix, due to specific interactions between the DNA and the solid phase, while contaminants do not interact with the solid phase to the same extent as DNA does, and thus may be separated from the sorbed DNA, e.g. by a washing step. Once the contaminants have been removed, the DNA has to be recovered from the solid phase by an eluting step, which usually includes a step of rinsing the solid phase with a solution (mobile phase) comprising compounds that minimize the interaction between the solid phase and the DNA, thus removing the DNA from the solid phase. The mobile phase comprising the DNA (eluate) is then collected. These solid phase-based methods enable an automation of the process of DNA isolation and purification. In addition, also rather minute amounts of DNA can be reliably processed using these methods.
Anion-exchange methods are based on the interaction between the negatively charged phosphates of the nucleic acids and positively charged surface molecules on the anion-exchange carrier (Forcic et al., J. Chromatogr. A 2005, 1065(1), 115-120). Under low-salt conditions DNA present in solution selectively binds to the stationary phase, and impurities such as RNA, cellular proteins, and metabolites may be washed away from the stationary phase using medium-salt buffers. In the next step, DNA can be eluted from the stationary phase using a buffer containing a high concentration of salt. The purified DNA is then recovered from the eluate by alcohol precipitation.
In silica-based methods, nucleic acids are selectively sorbed to a silica-gel membrane in the presence of high concentrations of chaotropic salts (Hanselle et al. Leg Med (Tokyo) 2003, 5 Supp. 1, S145-S149). RNA, cellular proteins, and metabolites are washed away from the membrane, and the DNA is then eluted from the silica-gel membrane using a low-salt buffer.
Also solid-phase methods based on the interaction between DNA and magnetic particles as a stationary phase are known in the state of the art (Prod{hacek over (e)}lalová et al. J. Chromatogr. A 2004, 1056, 43-48). Even though sorption methods allow the isolation of high-quality DNA, the number of steps to be carried out in these “bind-wash-elute” routines still is comparatively high and thus time-consuming. In addition, due to its excellent solubilizing properties SDS significantly influences the chromatographic behavior of a mixture of components to be separated in respect to the retention time, separation efficiency, etc., and even small variations of the SDS concentration in a sample may lead to a different chromatographic result. Furthermore SDS can bind to any anionic ion exchanging resin, either interfering with the binding of DNA or eluting together with DNA in the elution step.
For this reason a need exists for a fast, simple and reliable method of removing anionic surfactant ions in the presence of nucleic acid from a sample solution, wherein the nucleic acids, particularly the DNA essentially stay in solution. In addition, it should be possible to integrate this method of removing anionic surfactant ions in a method for isolating purified nucleic acids, in particular DNA and especially genomic DNA, from biological samples, such as tissue and blood, wherein the number of steps to obtain the purified nucleic acids is reduced in comparison to the known sorption procedures, such as anion-exchange and silica-based methods, without compromising the purity of the nucleic acids obtained. According to the present invention the term “nucleic acids” comprise any type or DNA or RNA as well as a mixture of DNA and RNA of any type. Particularly, dependent from the conditions and the steps used, either a mixture of DNA and RNA is obtainable or highly purified DNA can be prepared, which is as well separated from RNA. If in the following the term “DNA” is used, the DNA-containing purified nucleic acid sample is meant, either comprising RNA, or separated from RNA as well. Preferably the conditions of the method are resulting in a highly purified DNA, comprising essentially no RNA.
A detailed analysis of the known methods for isolating and purifying nucleic acids, in particular DNA, from cell-containing biological samples, i.e. methods for removing contaminants like anionic surfactant ions from the sample revealed that all these methods suffer from the fact that the nucleic acids do not remain in solution during the whole procedure of isolating and purifying. Instead, the nucleic acids either have to be precipitated or have to be bound, adsorbed or absorbed onto a solid matrix in the course of the isolation/purification procedure in order to remove the contaminants present in the cell lysate or introduced during the isolation and purification procedure. In consequence, additional steps of re-dissolving the nucleic acids from a precipitate or eluting them from a solid phase are necessary, which renders all of the methods mentioned above more or less time-consuming.
In preliminary experiments it has been found that concentrations of SDS above 312 μmol/L strongly inhibit PCR reactions. In order to have the possibility of directly using the sample in a subsequent PCR reaction, it would be useful to obtain a purified sample solution of alkaline or neutral pH in order to avoid the step of neutralizing the sample solution.
It was therefore an object of the present invention to provide a fast and efficient method for removing anionic surfactant ions at neutral or alkaline pH in the presence of nucleic acids, wherein the nucleic acids, in particular the DNA preferably stays essentially in solution during the removal procedure.
In the present invention it has surprisingly been found, that monovalent ions of alkali metals and/or divalent ions of alkaline earth metals selected from the group comprising, preferably consisting of Rb+, Cs+, Ca2+, Sr2+ and Ba2+, or a mixture thereof can be used for selectively precipitating anionic surfactant ions from a solution comprising such ions and nucleic acids, while the nucleic acids, particularly the DNA remains essentially in solution.
Accordingly, the present invention provides a method of selectively precipitating anionic surfactant ions from a liquid sample mixture comprising such ions and nucleic acids, comprising the steps of: 1. Adding to the sample mixture a solution (precipitating solution), comprising monovalent ions of alkali metals and/or divalent ions of alkaline earth metals selected from the group comprising, preferably consisting of Rb+, Cs+, Ca2+, Sr2+ and Ba2+, or a mixture thereof, and 2. optionally incubating the mixture comprising the sample mixture and the precipitating solution to ensure completeness of precipitate formation. In a preferred embodiment the precipitating solution comprises divalent ions of alkaline earth metals, in a particular preferred embodiment Sr2+-ions
Anionic surfactants according to the present invention are e.g. sulfates, sulfonates and carboxylates, preferably alkyl sulfates (fatty alcohol sulfates), alkane sulfonates, alkylbenzene sulfonates and alkyl carboxylates. Particularly preferred are surfactants providing surfactant ions showing a similar precipitation behaviour as dodecyl sulfate ions (DS−), more preferred are surfactants providing sulfate ions and most preferred are surfactants providing a source of dodecyl sulfate ions. As a source of dodecyl sulfate ions any compound releasing into solution dodecyl sulfate ions (H3C(CH2)11SO4−) upon dissolution in water may be used. The source of dodecyl sulfate ions preferably is selected from the group comprising sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate and lithium dodecyl sulfate, and most preferably is sodium dodecyl sulfate.
In terms of the present invention a precipitating solution is a solution comprising a water-soluble salt of alkali metals and/or alkaline earth metals, like for example RbCl, SrCl2, CaCl2 or BaCl2 which upon dissolution in water provides monovalent ions of alkali metals and/or divalent ions of alkaline earth metals. These ions are supposed to form a complex with anionic surfactant ions like e.g. dodecyl sulfate ions being insoluble in water, in aqueous solutions of neutral or alkaline pH, or in aqueous buffers, which results in precipitate formation, thus removing the dissolved surfactant ions from solution.
The concentration of the monovalent and/or divalent metal ions in the precipitating solution preferable is in the range of 0.1 to 10 mol/L, preferably of 0.5 to 5 mol/L, more preferably of 0.75 to 2.5 mol/L, and most preferably of 0.9 to 1.2 mol/L. Such a rather high concentration of metal ions in the precipitating solution is preferred in order to allow addition of a small volume and accordingly to avoid a too high dilution of the liquid sample.
The volume of precipitating solution added to a certain volume of sample mixture (lysate) depends upon the concentration of surfactant ions in this sample mixture. In a preferred embodiment, the volume ratio of liquid sample to so precipitating solution is in the range of 4:1 to 12:1, preferably 5:1 to 11:1, more preferably 6:1 to 10:1 and most preferably 7:1 to 9:1. The concentration of the surfactant ions in the liquid sample (lysate) preferably is approximately between 10 and 50 mmol/L. If for example 80 μL of a lysate comprising approximately 25 mmol/L dodecyl sulfate ions is used as a sample, then preferably 10 μL of a 1 M precipitating solution is added to precipitate the dodecyl sulfate ions in this sample.
In a preferred embodiment, the mixture obtained by adding the precipitating solution to the sample mixture is incubated at −10° C. to 10° C., preferably at −5° C. to 5° C., more preferably at −2.5° C. to 2.5° C. and most preferably at −1° C. to 1° C. after adding the precipitating solution to the sample mixture. The lower the incubation temperature, the faster precipitation is completed. However, the incubation temperature should be above the freezing point of the mixture to avoid a solidification of the whole mixture. The freezing point of a particular sample mixture depends upon the amount of salt dissolved in that mixture which is well known to a person skilled in the art, and the incubation temperature will be chosen accordingly. The step of incubating preferably is carried out for 3 to 60 min, preferably for 5 to 30 min and most preferably for about 10 min. Preferably the mixture is left to stand for the time given in an ice bath, which ensures completeness of precipitate formation. The precipitate may then be removed from the sample in a further step, preferably by centrifugation or filtration, more preferably by gel filtration chromatography. In case the precipitate is removed by any other procedure than gel filtration chromatography, the liquid remainder (e.g. supernatant after centrifugation or eluate after filtration) is further preferably purified by gel filtration chromatography.
For this purpose preferably the chromatographic device described in copending application with the title “chromatographic device and method for isolating and purifying nucleic acids” of the same applicant having the same filing date as the present application is used, which comprises at least one chromatographic unit, comprising: 1. A hollow body having an inlet and an outlet, the hollow body comprising a solid matrix providing size exclusion properties, preferably forming a gel bed; 2. a porous frit, filter, fleece or membrane, placed between the outlet and the solid matrix to retain the solid matrix within the chromatographic unit but preferably allowing nucleic acids of any size to pass, 3. a non-porous ring placed between the porous frit, filter, fleece or membrane and the matrix, sealing the outer area of the frit, filter, fleece or membrane, to prevent the mobile phase from entering the frit, filter, fleece or membrane without passing the matrix, 4. optionally at least one removable closing device to seal the inlet and/or outlet of the chromatographic unit, and 5. optionally at least one collection tube to collect the mobile phase (eluate) after having passed the matrix, wherein the solid matrix preferably is a gel forming polymer having a size exclusion limit of 150 to 500 base pairs (bp), preferably 200 to 400 bp, and most preferably 250 to 300 bp. Preferably the gel forming polymer has a corresponding size exclusion limit of 10 to 10000 KDa, more preferred of 20 to 8000 kDa.
This device allows a “negative” chromatography, where in contrast to the other chromatographic methods commonly used for the chromatographic purification of nucleic acids, not the nucleic acid, but the contaminants are sorbed to/retained on the solid matrix, thus allowing the chromatographic purification to be carried out in one single rinsing step. This chromatographic device does not only remove contaminants of small molecular weight, but also acts as a depth filter for solid material, in particular for the precipitate formed from surfactant ions and the monovalent alkali metal ions or divalent alkaline earth metal ions. Said precipitate does not enter the gel bed, but remain on its upper surface. This is even more surprising, since solid material usually tends to clock the pores of a gel, thus hampering or disturbing chromatography. Using this chromatographic device in combination with the method for precipitating surfactant ions of the present invention, samples containing highly purified de-salted nucleic acids, comprising in particular DNA and especially gDNA essentially free from residual surfactant ions can be obtained.
The chromatographic device is not limited to a special shape. Any device commonly used in chromatography may be used. The chromatographic device may be selected from, but not limited to, a traditional column used for suction or pressure column chromatography, a spin column, or a multiwell plate. In general, so-called chromatographic columns, having a circular cross-section, are used, whose diameter is small in comparison to their length. The column may for example be of cylindrical or conical shape or a combination thereof.
The chromatographic device described above is particularly suitable for size exclusion chromatography (SEC). If an organic solvent is used as the eluent (mobile phase), SEC is also called gel permeation chromatography (GPC). For purifying nucleic acids preferably a water-based mobile phase, such as water, an aqueous organic solvent or an aqueous buffer/solution is used as mobile phase. In this case SEC is also referred to as gel filtration chromatography. Size exclusion chromatography is a chromatographic method, wherein molecules are separated based on their size, or more precisely based on their hydrodynamic volume. Commonly, a solid matrix able to form a gel bed, when suspended in an aqueous medium, such as a dextran, agarose, polyacrylamide, or a mixture thereof, is suspended in a buffer and packed in the hollow body of a column made of glass, plastic, Teflon or any other material that neither reacts with the mobile phase nor the analyte. The sample to be purified is then applied to the center of the gel bed's upper surface, and allowed to pass through the gel, either by gravity or forced by centrifugation or pressure. Preferably centrifugal forces are applied to move the mobile phase down the column, wherein the columns are spun in a centrifuge (so-called spin column technique). Due to the cross-linking in the gel, pores of a certain size exist inside the gel. Small molecules are able to penetrate the pores, and therefore move through the gel bed more slowly, being retained as they pass down the column, while large molecules cannot penetrate the pores and move down the column more quickly. After having passed the column, the mobile phase (now referred to as eluate), containing the purified analyte, is then collected at the outlet of the column. To retain the solid matrix within the hollow body of the column, a porous frit, filter, fleece or membrane is preferably placed between the outlet of the column and the solid matrix.
In SEC, the size exclusion limit defines the molecular weight, where molecules are too large to be trapped in the stationary phase. The size exclusion limit of a solid matrix can be adjusted by the degree of cross-linking in the gel. A wide variety of solid matrices able to form a gel bed with different degrees of cross-linking are commercially available.
A problem often encountered in gel filtration chromatography, and particular in gel filtration chromatography using spin columns, is that the mobile phase may run down along the inner wall of the column, thus entering the frit, filter, fleece or membrane without having passed the solid matrix. This is especially true for high-throughput applications, when not all of the sample mixture to be purified is applied exactly to the center of the gel bed's flat surface, or the sample is applied to quickly. When the mobile phase does not enter the gel bed, no chromatographic separation occurs, and a contaminated eluate is obtained. To overcome this problem, the chromatographic device preferably is equipped with a non-porous ring placed between the porous frit, filter, fleece or membrane and the matrix. This ring seals the outer area so of the frit, filter, fleece or membrane, thus preventing the mobile phase from entering the frit, filter, fleece or membrane without having passed the matrix. In addition, the velocity of the mobile phase inside the column is slowed down, thus improving selectivity.
Optionally the chromatographic device may contain at least one removable closing device to seal the inlet and/or the outlet of the chromatographic unit. If both the inlet and the outlet are equipped with such a removable closing device, the closing devices used to seal the inlet and those used to seal the outlet may be the same or different.
The chromatographic device may furthermore comprise at least one collection tube to collect the mobile phase (eluate) after having passed the matrix. It may further be equipped with one collection tube per chromatographic unit, i.e. if the chromatographic device only contains one chromatographic unit, preferably only one collection tube will be comprised. If on the other hand the chromatographic device contains several chromatographic units for example 24, 48 or 96 chromatographic units in form of a multiwell plate, then also more than one collection tube will be comprised, preferably in form of a multiwell plate, too. Additional collecting tubes may be supplied for collecting the liquid drained from the column during pre-spinning.
The gel-forming polymer preferably is selected from the group of comprising dextrans, agarose, polyacrylamide, or mixtures thereof, and more preferably is a mixture of a dextran and a polyacrylamide. Such gel-forming polymers of different size exclusion limits are commercially available, for example under the trademark name of Sephacryl, Sephadex, or Sepharose. A particular preferred solid matrix is the S-400 HR Sephacryl resin, commercially available from GE-Healthcare, which is a spherical allyldextran/N,N′-methylene bisacrylamide matrix with a size exclusion limit of 271 bp (corresponding to 20-8000 kDa). Further suitable gel-forming polymers may have a methacrylic basis, like a hydroxylated methacrylic polymer, for example Toyopearl HW 65 available from Tosoh Bioscience LLP (former Toso-Haas) with a size exclusion limit of 40 to 5000 kDa.
The removable closing device preferably may preferably represent a disposable closing device, selected from the group comprising lit foils, seals and break-away ends, or a re-closable closing device, selected from the group comprising of screw caps and snap-on caps. If both, the inlet and the outlet of the chromatographic unit, are sealed with the removable closing device, the solid matrix may be supplied in the form of a gel, preswollen in a solvent selected from the group comprising water, the homogenous mixtures of organic solvents with water, or aqueous buffers inside the closed chromatographic device. In this case, the solvent is preferably purged from the chromatographic unit, while simultaneously establishing the matrix bed by centrifugation immediately prior to use (pre-spinning).
The chromatographic device may also comprise a plurality of chromatographic units in a parallel fashion, preferably in the form of a multiwell plate wherein each well of the multiwell plate contains one separate chromatographic unit.
The precipitate comprising the solid material, formed from the interaction of surfactant ions and the ions of alkali metals or alkaline earth metals, as well as further solid material, being insoluble in the lysis buffer, is removed before or in the gel filtration step, for example complexed dodecyl sulfate ions or derivatives thereof, precipitated proteins and the like. A further advantage of the method of present invention is that due to the use of size-exclusion chromatography instead of simple filtration or centrifugation step, not only solid material is separated from the dissolved nucleic acid. In addition, also dissolved contaminants such as proteins, residual small fragments of nucleic acids, metabolites, lipids and other components typically being present in biological samples are removed in one single step.
Gel filtration chromatography preferably is carried out using the chromatographic device described above and comprises the following steps: 1. establishing the matrix bed in the chromatographic unit by centrifugation (pre-spinning), 2. applying the sample comprising the dissolved nucleic acids and precipitate formed from surfactant ions like e.g. dodecyl sulfate ions and alkali or alkaline earth metal ions to (the center of) the matrix bed's upper surface and finally 3. eluting the nucleic acids from the chromatographic unit by centrifugation and simultaneously collecting the eluate.
The step of pre-spinning preferably is carried out by centrifuging the device at 500 to 900×g for 1 to 7 min, preferably at 700×g for 2 to 5 min, and most preferably at 700×g for 3 min.
The volume of the matrix bed in the chromatographic unit preferably is in the range of 100 μL to 2 mL, more preferably in the range of 500 μL to 1 mL, and most preferably is 700 to 800 μL. The matrix is preferably provided as a dispersion of the gel-forming polymer in water, a salt solution, e.g. 0.9% NaCL, or a suitable buffer, like e.g. TE, TAE, PBS or similar or in diluted buffers, whereas said dispersion comprises preferably 60-90%, more preferably 70-80% and in particular 75% of the gel-forming polymer. The filling level of the matrix bed in the chromatographic unit preferably is in the range of 0.5 cm to 2.0 cm, more preferably 1.0 to 1.5 cm. In a standard 96-well plate the volume of the matrix bed preferably is about 0.8 mL. The exact volume and the filling level of matrix bed used depend on the size and shape of the hollow body defined by the column, as well as on the kind and amount of the sample to be purified, which is well known to a person skilled in the art. Preferably a sample volume of up to 100 μL is applied to a column packed with 600 μL to 800 μl or a filling level of 1.0 cm to 1.5 cm of matrix bed.
The step of eluting the DNA from the chromatographic unit preferably is carried out by centrifuging the device at 500 to 900×g for 1 to 7 min, preferably at 700×g for 2 to 5 min, and most preferably at 700×g for 3 min. Preferably the time and centrifugation force which is applied should correspond to the time and force used for pre-spinning (see above).
Even though the method of selectively precipitating surfactant ions of the present invention may be applied to any sample mixture comprising surfactant ions and nucleic acids, the liquid sample preferably is the lysate obtained from a cell-containing biological sample by treating the sample with a lysis buffer. A lysis buffer is an aqueous solution, comprising active components such as detergents (surfactants), to disrupt and/or break the cellular membrane of a cell, causing the intracellular components, such as DNA, RNA, proteins, lipids, metabolites etc., to be released into solution. The obtained mixture comprising the former intracellular components is called lysate. Preferably a lysis buffer is used which allows a fast lysis procedure under low-salt lysis conditions, as a high amount of salt in the lysate additionally complicates the purification process. It is further advantageous if the other components of the lysis buffer do not interfere in any of the subsequent steps of the method of the present invention or subsequent down-stream applications, or that potentially interfering components can be removed by a simple and fast purification step, while the nucleic acids, in particular the DNA remains essentially in solution.
In a particular preferred embodiment the sample mixture preferably is the lysate obtained from a cell-containing biological sample by treating the sample with a lysis buffer as described in copending application with the title “method for isolating and purifying nucleic acids” of the same applicant having the same filing date as the present application, which comprises a source of surfactant ion, preferably sulfate ions, particularly preferred dodecyl sulfate ions (DS−), but is preferably essentially free of any complexing or chelating agents like e.g. ethylenediamine tetraacetic acid (EDTA). The lysis buffer preferably comprises a buffering substance, H2504 and a source of surfactant ions, has a pH of 7.5 to 10, preferably of 8 to 9 and most preferably of 8.5, but preferably is essentially free of any complexing or chelating agent and Mg2+-ions. This buffer allows a fast lysis of sample material under low-salt lysis conditions, i.e. hypotonic conditions, wherein the total ion concentration in the buffer solution is lower than the total ion concentration within the cells to be lysed. In the case of NaCl, for example, an aqueous solution comprising less than 0.9 wt % NaCl (about 155 mmol NaCl, corresponding to about 310 μmol/L of dissolved ions) is hypotonic. Using this buffer, even samples containing a rather high amount of solid material, for example tissue samples, are usually completely lysed within less than 40 min at e.g. 56° C. Lysis can be carried out at temperatures ranging from 45° C. to 70° C., preferably from 50° C. to 68° C., and most preferred at 62° C., dependent from whether it is preferred that RNA remains in the isolated nucleic acids or not. A simple method for disintegrating the RNA in the sample is by heating the sample to a temperature of at least 60° C. without any further addition of an disintegrating agent. If the RNA shall remain in the sample heating of the sample only up to 58° C., preferably up to 56° C. is recommended.
Increasing the temperature during or after the lysis step up to 80° C. additionally denaturates the proteins (e.g. enzymes) in the sample without affecting the desired DNA, in particular genomic DNA. In this case of course RNA is not obtained.
Since this buffer is essentially free of any agent acting as a complexing or chelating agent for the divalent ions necessary as co-factors for the polymerase in PCR reactions, and the pH of the lysis buffer falls within the optimum pH range for PCR reactions, the lysate obtained using this lysis buffer can be directly used in downstream applications such as for example qRT-PCR, after removing the surfactant ions according to the method of the present invention.
As mentioned above any anionic surfactant usable as a detergent for disrupting cells can be contained in the lysis buffer. The most commonly used detergents for said approach are dodecyl sulfate ions providing surfactants. Any compound releasing into solution dodecyl sulfate ions (H3C(CH2)22SO4−) upon dissolution in water may be used as a source of dodecyl sulfate ions. The source of dodecyl sulfate ions preferably is selected from the group comprising sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate and lithium dodecyl sulfate, and most preferably is sodium dodecyl sulfate.
According to the present invention the lysis buffer preferably is essentially free of a chelating or complexing agent. Non-limiting examples for such agents are EDTA, EGTA , EDDS (ethylene diamine diacetic acid), NTA (nitrilo triacetic acid), gluconic acid, isoascorbic acid, tartaric acid, citric acid, iminodisuccinate, triethanolamine. In terms of the present invention a lysis buffer is essentially free of a chelating or complexing compound, e.g. EDTA, if it contains less than 10 mg/L, preferably less than 1 mg/L, more preferably less than 0.1 mg/L, even more preferably less than 0.001 mg/L, and most preferably the lysis buffer of the present invention does not contain any EDTA at all (0 mg/L).
The lysis buffer may be used in combination with a protease. A protease, sometimes referred to as proteinase, is an enzyme catalyzing the hydrolytic cleavage of the peptide bonds linking the amino acids in a polypeptide chain (protein). Suitable proteases are known from the state of the art and include QIAGEN Proteinase K or QIAGEN Protease, (QIAGEN, Hilden, Germany) without being limited to them. It has been found that the rather expensive Proteinase K may be replaced by the cheaper QIAGEN Protease without compromising the result of the purification procedure. The protease may already be present in the ready-to-use lysis buffer solution as supplied or may be added to the mixture by the user after mixing the biological sample with the lysis buffer.
The concentration of the source of anionic surfactant ions in the buffer depends on the sample to be lysed. The concentration of the source of surfactant ions in the buffer preferably is 1 to 100 mmol/L, more preferably 5 to 75 mmol/L, even more preferably 10 to 50 mmol/L and most preferably 25 mmol/L. The buffering substance can be any suitable buffering substance providing a pH of at least 7.5 like e.g. TRIS, HEPES, HPPS or any ammonia buffer. The preferred buffering substance is TRIS. The concentration of the buffering substance in the buffer preferably is in the range of 1 to 100 mmol/L, more preferably 5 to 75 mmol/L, even more preferably 10 to 50 mmol/L and most preferably 25 mmol/L. The molecular ratio of the buffering substance, e.g. TRIS to the source of surfactant ions in the buffer preferably is in the range of 3:1 to 1:3, more preferably 2:1 to 1:2, even more preferably 1.2:1 to 1:1.2 and most preferably is 1:1.
If samples comprising a high amount of liquid compounds such as blood are lysed, preferably a double concentrated buffer (2×) is used, wherein the concentration of each the source of surfactant ions and buffering substance like TRIS is 2 to 200 mmol/L, more preferably 10 to 150 mmol/L, even more preferably 20 to 100 mmol/L, and most preferably 50 mmol/L, respectively, to avoid too high dilution of the mixture by the sample liquid. The lysis buffer may also be provided in form of a concentrate to be diluted by the user prior to use, comprising for example the ten-fold concentration of the source of surfactant ions and buffering substance (10× lysis buffer).
The lysis buffer may comprise further active components selected from the group comprising stabilizers such as sodium azide, solubilizing agents or the like, which are well known to a person skilled in the art.
H2SO4 is used as acidic compound in the buffer composition due to the rather low solubility of sulfate salts, for example SrSO4, in order to ensure an effective removal of any excess alkali or alkaline earth metal ions from the precipitating solution. The concentration of chloride ions in the buffer preferably is less than 10 mmol/L, more preferably less than 1 mmol/L, even more preferably less than 0.1 mmol/L.
Using the lysis buffer described above lysis of cell-containing biological samples preferably is carried out by a method comprising the fowling steps: 1. mixing the sample with a lysis buffer described above, 2. incubating the mixture obtained in step 1 to obtain a lysate comprising DNA, RNA and proteins.
The volume of lysis buffer used to lyse a certain amount of sample material depends upon the kind and size of the sample material being processed. In the case of tissue samples it may for example be useful to cut large tissue sample into smaller pieces of approximately 4 mm3 or less, before mixing the sample with the lysis buffer. Preferably a lysis buffer volume of 20 to 150 μL, more preferably of 30 to 120 μL, even more preferably of 50 to 100 μL and most preferably of 80 μL is used for the lysis of 10 mg of sample tissue.
If the nucleic acids to be isolated are desoxyribonucleic acids, RNA present in the lysate may be optionally disintegrated after lysing the sample, but before precipitating the surfactant ions. The term “disintegrating RNA” comprises any method of reducing the amount of dissolved RNA in the lysate and/or inactivating the RNA and/or facilitating its separation from the DNA, including any method of thermally, chemically and/or enzymatically hydrolyzing, digesting, transforming and/or decomposing RNA, either partially or completely, and/or removing the RNA or its fragments from the solution, e.g. by precipitation, sorption procedures or the like. Lysing the biological sample by incubating a mixture of the sample and the lysis buffer, and the optional step of disintegrating the RNA present in the lysate may preferably be carried out in a single step, preferably by heating the mixture to a temperature equal to or above 60° C., more preferably to 60° C. to 70° C., even more preferably to 61° C. to 65° C., and most preferably to 62° C. The period of time a sample has to be heated in order to ensure complete lysis and RNA disintegration depends on the kind and amount of sample being processed. Preferably the mixture is heated for 10 to 80 minutes (min), more preferably for 15 to 60 min, even more preferably for 20 to 50 min, and most preferably for 30 to 45 min. If an increase of the temperature up to 80° C. is carried out to denaturate the proteins as described above, RNA of course is disintegrated as well.
Using the method for selectively precipitating anionic surfactant ions of the present invention in combination with the lysis procedure, the optional step of disintegrating RNA present in the lysate and the chromatographic step described above, high-quality nucleic acids, particularly highly purified DNA, and in particular even high-quality genomic DNA, can be obtained from a variety of biological samples. In this case, a biological sample preferably is processed by o the following steps: 1. mixing the sample with a lysis buffer comprising a source of anionic surfactant ion, preferably dodecyl sulfate ions (DS−), but being essentially free of a complexing or chelating agent, 2. incubating the mixture obtained in step 1 to obtain a lysate comprising at least DNA, RNA, and proteins, 3. optionally disintegrating the RNA present in the lysate, 4. precipitating the surfactant ions from the lysate according to the method of the present invention, 5. separating the nucleic acids from the precipitate and/or further contaminants present in the lysate preferably by size-exclusion chromatography or any known alternative method for separating the nucleic acids from the precipitate to obtain a purified DNA-containing eluate. In this method, the nucleic acids, in particular the DNA remain essentially in solution during all of steps 2 to 5.
In this way purified nucleic acids, particularly highly purified DNA can be obtained from tissue samples in about only 45 min (30 min lysis, 10 min of precipitation, 3 min pre-spinning of the column and 3 min for the chromatographic separation itself), while approximately 2.5 h typically are necessary for the lysis and purification of the same amount of tissue using e.g. the QIAamp kit (QIAGEN, Hilden, Germany).
The quality and purity of the nucleic acids isolated by this method is equal, or in many cases even superior, to the quality and purity of nucleic acids obtained by state of the art methods for bench-scale purification, such as for example the very successful QIAamp technology (QIAGEN, Hilden, Germany) with respect to purity and yield as judged by UV/Vis spectroscopy, gel electrophoresis, conductivity measurements, HPLC analysis, PCR and further assays. In addition, the DNA-containing eluate obtained by this method may be frozen for longtime storage or may be processed in downstream application like quantitative real time PCR (qRT-PCR), PCR and the like, immediately after chromatography without the need for any additional steps to isolate the nucleic acids from the eluate. As the nucleic acids, in particular the DNA remain essentially in solution during the purification process, and neither is precipitated by the addition of organic solvents such as for example ethanol, nor is sorbed or bound to a solid matrix such as a silica-membrane or an anion exchange resin, the method described herein is much faster than the methods for isolating and purifying nucleic acids from the step of cell lysis to the purified DNA sample known from the state of the art. Furthermore, the whole method can be fully automated.
In principle all kind of nucleic acids (RNA and DNA) can be isolated and/or purified from a wide variety of biological samples, including synthetic, genetically engineered or naturally occurring single-stranded or double-stranded DNA, oligo- and polynucleotides of desoxyribonucleotides or ribonucleotides, fragments of DNA obtained by partly digesting DNA using restriction endonucleases, mitochondrial DNA, plasmid DNA, and metagenomic DNA, representing the entirety of DNA obtained from all microorganisms found in a biotope or a biocenosis.
The method described herein is even suitable for isolating and purifying genomic DNA, which is the high molecular weight DNA obtained from one single organism, comprising the entirety of genetic information of this organism, in contrast to plasmid DNA, DNA partly digested by the action of restriction endonucleases, and metagenomic DNA. If the precipitate is separated from the sample as described above, not only the solid material is retained on the upper surface of the column, but also smaller fragments of DNA are retained within the chromatographic material. Due to its high molecular weight and large size, intact high quality genomic DNA is difficult to isolate and purify, as a comparably high risk of degradation of genomic DNA exists, either by mechanical stress during the isolating procedure, in particular sheer stress, or by chemical and enzymatic degradation. Degraded DNA, on the other hand, may lead to both quantitative and qualitative errors in downstream analyses. The method of the present invention provides a fast, robust, safe, easy-to-handle and yet mild method for purifying DNA, in particular genomic DNA, from a variety of different biological samples by precipitating the contained surfactant ions, like dodecyl sulfate ions.
Using the method of the present invention nucleic acids, preferably DNA, more preferred genomic DNA present in the lysates obtained from a wide variety of starting materials can be purified, including, but not limited to, animal and human tissue, for example liver, spleen, lung, heart, brain, kidney etc., animal and human blood, liquor, sputum or sperm, cell cultures of animal and human cells, animal and human bone marrow, yeast, bacteria, insects, plants, and rodent tails. Preferably, the sample is a lysed cell-containing biological sample of animal or human origin. In another preferred embodiment, the samples comprise Gram-negative bacteria. The sample may have been lysed immediately after having been taken from its natural environment (fresh sample), or may have been stabilized by freezing or by the action of chemical stabilizing agents, such as for example formalin-fixation and paraffin-embedding (FFPE tissue) or blood stabilizing agents comprising citrate or Heparin, before it has been lysed. Even more preferably the sample is a lysed sample selected from the group comprising fresh or frozen tissue and blood, most preferably from mammalian tissue and blood.
If the method for precipitating according to the present invention is used in combination with the lysis procedure, the optional step of disintegrating RNA present in the lysate and the chromatographic step described above the amount of DNA obtained from 10 mg of a sample usually around 5 to 70 μg genomic DNA are obtained. The specific amount of DNA depends upon the sample, for example its kind and age, which is well known to a person skilled in the art.
Amounts of about 10 mg, based on the original amount of cell-containing biological sample before cell lysis, are the amount of sample commonly analyzed in molecular diagnostics. It should, however, be understood, that using the method of the present invention, it is also possible to process larger or smaller amounts of sample material, e.g. in the g-range or μg- to ng-range, respectively. In this case, the amounts of reagents, buffers, solid matrix as well as the dimension of the chromatographic device have to be adjusted by up- or down-scaling, which is well known to a person skilled in the art.
The invention further provides a kit for the isolation and purification of nucleic acids, preferably comprising genomic DNA, comprising: 1. A source of monovalent ions of alkali metal and/or divalent ions of alkaline earth metal selected from the group comprising, preferably consisting of Rb+, Cs2+, Ca2+, Sr2+, Ba2+ or mixtures thereof, either in the form of a water-soluble alkaline earth metal salts to be dissolved by the user, or as a stock solution to be diluted by the user, or as a ready to use solution and one or more components selected from the group comprising, preferably consisting of 2. a lysis buffer comprising an anionic surfactant, preferably the lysis buffer described above, 3. a chromatographic device for gel filtration chromatography, preferably the chromatographic device described above, and 4. optionally one or more primers for the amplification of one or more target nucleic acids. Further the kit preferably comprises instruction for presently described isolation and/or purification method.
Materials and General Experimental Procedures
Gel filtration media were obtained from GE-Healthcare (Freiburg, Germany), ion-exchange media were obtained from Merck KgaA (Darmstadt, Germany).
Unless otherwise noted, the tissue samples analyzed were rat liver tissue samples.
Determination of the amount and purity of gDNA: To estimate the amount of gDNA (gDNA yield) in a purified sample (eluate), the absorbance of the sample was measured at a wavelength of 260 nm by UV/Vis spectroscopy. A background absorption value, measured at 320 nm was subtracted from the OD260 value (optical density at 260 nm), and the value was multiplied by 50, the specific absorbance factor of DNA, and by the dilution factor to obtain the gDNA concentration in μg/μL. In addition, UV/Vis spectroscopy was also used to judge the purity of the DNA obtained. Residual solid particles do not exhibit a distinct absorbance peak, but lead to an elevated baseline in the whole spectrum. Free haemoglobin has an absorbance maximum at a wavelength of 410 nm, while salts and preservatives like sodium azide absorb at a wavelength below 230 nm. A Spectramax II (Molecular Devices, Sunnyvale, Calif., USA) 96-well plate photometer was used to record the UV/Vis spectra.
A more precise determination of the amount of gDNA obtained was carried out using HPLC analysis. The area under curve (AUC) for the gDNA-containing peaks in the spectra was calculated by the software and compared to a HPLC standard curve, to determine the amount of gDNA in a sample. HPLC analysis was also used to determine the purity of the samples using a Vision BioCad workstation (Perseptive Biosystems, Framingham, Mass., USA). A 0.83 mL Peek column filled with the ion exchange resin TMAE-Fractogel(S) (E. Merck, Darmstadt, Germany) was used. The samples were analyzed at a flow rate of 1.5 mL/min in an increasing CaCl2 gradient, starting from 0 mmol/L to 300 mmol/L over a period of 35 column volumes, buffered at pH 7.2. The absorbance was continuously monitored at 260 nm and 410 nm.
Agarose gel electrophoresis was carried out using a 50 mL 0.8% agarose gel, containing 2.5 μL SYBR-Green II. Samples were run using a voltage of 100 Volt for a time period of 40 min. The gels were analyzed using commercially available equipment from BioRad or LTF-Labortechnik (Wasserburg, Germany).
SDS quantification: The residual SDS concentration was determined by UV/Vis spectroscopy according to a modified procedure of Rusconi et al. adapted to be used within a 96 well photometer (Rusconi et al. Anal. Biochem., 2001, 295(1), 31-37). The assay is based on a specific reaction of the carbocyanine dye “Stains AII” (4,5,4′,5′-Dibenzo-3,3′-diethyl-9-methylthiocarbocyaninebromide) with SDS, which leads to the formation of a yellow colour (absorbance maximum at 438 nm). As SDS was used as the source of dodecyl sulphate ions in the present examples, it should be understood that the amount (molarity) of SDS in a solution equals the amount (molarity) of dodecyl sulfate ions present in solution.
1 mL of a stock solution of the dye (1.0 mg “Stains AII” in 1.0 mL 50% isopropanol) was diluted with 1.0 mL formamide and 18 mL water to obtain a ready-to-use solution of the dye. To determine the amount of SDS in a sample, 5 μL of the liquid sample were placed into a microtiter plate, mixed with 100 μL of the ready-to-use solution, and incubated at room temperature for 5 min in the dark before reading the plate at 438 nm. The amount of SDS in the sample was retrieved by comparison with a calibration curve, established by recording the absorbance of solutions containing a SDS concentration of 250, 167, 111, 74, 49, 32 and 21 μmol/L, respectively, at 438 nm.
Conductivity measurement: To determine the ion strength in the samples, conductivity measurements were carried out using a Consort C831 Conductometer (LTF-Labortechnik, Wasserburg, Germany), calibrated to 20° C. A minimum volume of 2 mL is necessary for the measurement, therefore aliquots of 20 μL of each sample were diluted with 1980 μL water prior to the measurement.
PCR amplifications: Real time-PCR (qRT-PCR) assays were performed on a Rotor-Gene 2000 or 3000 cycler (Corbett, Sydney, Australia) on a 50 μL scale, or in a TaqMan 7700 analyzer (Applied Biosystems, Foster City, Calif., USA).
For the jun RT-PCR assay, a commercially available kit (Part. No: 4327113F), based on a primer/probe system (FAM) from Applied Biosystems (Darmstadt, Germany), including a 20×Jun PCR primer/probe mix was used in combination with a 2×TaqMan PCR universal master mix from Applied Biosystems.
A genomic DNA standard was purified from rat tail using the QIAsymphony DNA kit on the QIA-symphony platform (QIAGEN, Hilden, Germany), and was further purified by subsequent anion exchange chromatography (AEX) using a QIAGEN tip 2500 according to the manufacturers' protocol (QIAGEN, Hilden, Germany). The gDNA was stored in aliquots at −20° C. and thawed immediately prior to use.
Lysis buffers of the following compositions were prepared: reference buffer A: 100 mmol/L TRIS, 5 mmol/L EDTA, 100 mmol/L MgSO4 and 100 mmol/L SDS, adjusted to pH 6.0 by the addition of H2SO4; reference buffer B: 100 mmol/L TRIS, 5 mmol/L EDTA, 100 mmol/L MgSO4 and 100 mmol/L SDS, adjusted to pH 8.0 by the addition of H2SO4; lysis buffer to be used in the method of the present invention (buffer C): TRIS 25 mmol/L, SDS 25 mmol/L, adjusted to pH 8.5 by the addition of H2SO4 (25% v/v). All buffer compositions were prepared as aqueous solutions. Reference buffer compositions A and B are based on standard buffer compositions, commonly used for lysing cell-containing material, additionally containing Mg2+-ions, which were reported to degrade RNA at alkaline pH and temperatures above 37° C. (N. G. AbouHaidar and I. G. Ivanov Z. Naturforsch. 1999, 54 c, 542-548). Samples of pork liver tissue (25 mg each) were incubated with 500 μL of reference buffer A, reference buffer B and the buffer according to the present invention, respectively, at 56° C. To aid depletion of proteins, 10 μL of QIAGEN Proteinase K (2.5 AU/ml) (QIAGEN, Hilden, Germany) was added to each liquid sample. While lysis of the pork liver tissue was usually complete within 40 min using reference buffer B and buffer C, residual tissue fragments were still present even after two hours of incubation when using reference buffer A. Thus, the results described by AbouHaidar et al. could not be verified.
Although it is known in the state of the art that potassium salts of dodecyl sulfate have a very low solubility at acidic or almost neutral pH, it was initially attempted to precipitate the dodecyl sulfate ions in the lysate obtained using reference buffer B by the addition of potassium ions. With the intention to directly use the lysates in a PCR reaction, after precipitation of dodecyl sulfate ions, and subsequent removal of the precipitate formed, solutions of alkaline potassium salts were added to the lysate in order to precipitate dodecyl sulfate ions, as PCR reactions usually require a pH of about 8 to 9.
In a first experiment 45 μL of a 0.25 M aqueous solution of KOH were added to the lysate (500 μL). The precipitate was removed by centrifugation for 5 min at 20,000×g in a HERAEUS Biofuge. An aliquot of the supernatant was analyzed by gel electropheresis on an agarose gel. The genomic DNA in the supernatant was found to be completely degraded, most probably due to the strong alkaline pH found in the sample after addition of potassium hydroxide. A titration experiment, wherein increasing amounts of an aqueous KOH solution were added to the lysate, revealed that KDS begins to precipitate at pH 10.9, but even at pH 12 dissolved SDS and its magnesium salt are still present within the sample. Accordingly, KOH cannot be used to precipitate dodecyl sulfate ions from a lysate comprising genomic DNA without degrading the genomic DNA.
For this reason potassium carbonate (K2CO3) and potassium bicarbonate (KHCO3) were tested as an alternative alkaline source of potassium ions. Samples of 20 mg of pork liver tissue were suspended in 500 μL of reference buffer B (pH 8.0) and incubated at 56° C. for 30 min. To precipitate the dodecyl sulfate ions from the lysate, 140 μL of a 0.25 M aqueous solution of K2CO3 per sample and 350 μL of a 0.25 M aqueous solution of KHCO3 per sample were added to two samples, respectively. All samples were incubated in an ice bath for 5 min. While the addition of KHCO3 did not alter the pH of the lysate significantly, a slight increase of pH was observed after addition of K2CO3. For this reason samples treated with K2CO3 were neutralized by adding 3 μL 2% HCl (aq). All samples were centrifuged to remove the precipitate, and aliquots of the supernatants (4 μL) were analyzed on an agarose gel. As a positive control DNA from two further samples of pork liver tissue lysed using reference buffer B was purified on a QIAsymphony platform (QIAGEN, Hilden, Germany) according to the QIAsymphony DNA handbook May 2008, using a bind-wash-elute concept based on magnetic particles including a RNAse treatment.
The electrophoresis gel is shown in
In further experiments the optimum composition of the lysis buffer as well as the standard optimum conditions for lysis and simultaneous disintegration of RNA present in the lysate using this lysis buffer were determined. The optimum buffer composition should comprise TRIS and SDS, both at a concentration of 25 mmol/L, the pH of the buffer solution being adjusted to pH 8.5 by the addition of sulfuric acid, but should be free of Mg2+-ions and EDTA. A protease such as QIAGEN Protease or QIAGEN Proteinase K (10 μL; 2.5 AU/ml) may be added to the buffer, preferably after resuspension of the tissue material to aid disintegration of proteins. Lysis preferably should be carried out at a temperature of 62° C. for 30 min. Unless otherwise noted, these lysis conditions were used in the following.
Different amounts of a 0.5 M aqueous solution of SrCl2 (25 μL and 50 μL, respectively) were added to lysates, obtained from lysis of 10 mg of tissue by incubating the samples in a lysis buffer C containing TRIS and SDS, both at a concentration of 25 mmol/L, adjusted to pH 8.5 by the addition of H2SO4. After having added the precipitating solution, the mixtures were incubated at room temperature or in an ice bath (ca. 0° C.) for 10 min. The precipitates formed were then removed by centrifugation or by filtration on a short gel filtration column filled with 600 μL of GE-Healthcare S1000 SF resin (GE-Healthcare, Freiburg, Germany), respectively.
Results are presented in
Example 3 revealed that filtration is more effective than a simple centrifugation in removing the precipitate formed after addition of SrCl2-solution. Therefore, the minimum amount of resin necessary for gel filtration chromatography was determined using the GE-Healthcare S1000 SF matrix. The results were compared to other filtration methods, in particular filtration through a QIAshredder column (QIAGEN, Hilden, Germany), a silica frit (Mat. No: 1016844, QIAGEN, Hilden, Germany), a bed of silica particles (QiaExII, QIAGEN, Hilden, Germany), a sterile filtration through a 0.2 μm membrane of a MiniSart filter (Satorius, Göttingen, Germany), and a commercially available G25 spin column (GE-Healthcare).
10 μL of a 1 M SrCl2-solution were added to 80 μL of lysates obtained as described in example 3. The mixture was incubated in an ice bath, and then applied to the different filtration devices. The mobile phase was moved through the filtration device using centrifugation, and the eluates obtained were collected and analyzed. Each experiment was carried out in duplicate. For each eluate, the amount of purified gDNA present was determined as described in the general methods. The amount of SDS was determined by UV/Vis spectroscopy according the protocol described in the general methods. Furthermore, the presence of ions in the eluates was determined by conductivity measurements according the general method. The results are presented in
As can be seen from
Using the S1000 resin, resin amounts of 400 μL or less were not sufficient to form a homogenous gel bed during centrifugation using a fixed angle rotor. The minimum amount of resin necessary to ensure homogenous gel bed formation in a spin column having a column height of 2.5 cm, and an inner diameter of 0.8 to 0.9 cm, was 600 μL.
A problem commonly observed in gel filtration chromatography using spin columns is the fact, that lysate may run down the inner walls of the spin column passing the frit , filter , fleece or membrane without having entered the gel bed. This is in particular true, when rather large sample volumes are applied to a column, or when the sample is not applied strictly to the center of the gel bed. In consequence, the contaminants are not removed in these samples. This problem has been overcome by introducing a non-porous ring, placed between the porous frit, filter, fleece or membrane and the matrix, sealing the outer area of the frit, filter, fleece or membrane, to prevent the mobile phase from entering the frit, filter fleece or membrane without passing the matrix.
In a next step the optimum size exclusion limit of the matrix bed was determined. 600 μL of different commercially available gel filtration matrices (Sephacryl resins comprising a matrix of spherical allyl dextran and N,N′-methylene bisacrylamide) were filled into a spin column as described above, equipped with a dense silica frit (Mat. No. 1017499, QIAGEN, Hilden, Germany), a plastic ring, and a screw cap. The size exclusion limits of the resins used (in base pairs, bp, or in kDa, respectively, as far as provided) are given in table 1.
All resins were equilibrated in water prior to use: 10 mL of the resin were mixed with 40 mL of water, the resin was allowed to settle, until a sediment of 10 mL was obtained, and the supernatant (water) was discarded. This procedure was repeated three times. Finally, the volume of the suspended resin was adjusted to 10 mL by the addition of water. The columns were prepared for use by establishing the gel bed in a pre-centrifugation step (pre-spinning) at 700×g for 5 g for 3 min. To each column the whole lysate, obtained from 10 mg tissue as described above, including the precipitate, was applied to the center of the gel bed's upper surface. The DNA was then eluted by spinning the column at 700×g for 3 min. Each experiment was carried out at least in duplicate. The amount of gDNA and SDS present in the eluates were measured as described above. The conductivity of the samples was determined using a diluted solution of a pooled sample, wherein all samples purified using the same resin were combined. The results are depicted in
To evaluate the extent of protein removal in the purification process and to determine the residual amount of protein present in the eluate obtained after gel filtration chromatography, samples purified according to example 4 using a S400 HR resin were analyzed by PAGE analysis on a commercially available TRIS/HEPES 4-20% gradient polyacrylamide gel (LTF-LABORTECHNIK), containing SDS. The gel was run at 100 volt for 25 min in the supplied TRIS/HEPES buffer using the NOVEX XCell II system (Invitrogen GmbH, Karlsruhe, Germany). 15 μL of the samples spiked with a protein size marker were diluted with 15 μL of the 2× buffer, provided with the gel. The mixture was heated to 95° C. for 5 min, then cooled in an ice bath, and afterwards loaded into the gel pocket. The gel was stained overnight by immersing it into Gradipure stain (Gradipore, Sydney, Australia), and then rinsed with water. The results are presented in
To detect minute amounts of residual contaminants such as proteins and RNA fragments, an anion exchange (AEX) HPLC analysis was performed on an TMAE-Fractogel S HPLC column using a CaCl2 gradient at pH 7.2 at a flow rate of 1.5 mL/min. A gradient ranging from 0 mmol/L CaCl2 to 300 mmol/L CaCl2 in water, comprising 5% TRIS (pH 7.2), over 35 column volumes was established. As an injection volume 200 μL were used.
In a first experiment 200 μL of the crude lysate obtained from incubating 10 mg pork liver tissue with the buffer described above in the absence of RNase were analyzed. Seven fractions were collected from the HPLC, reacted with the dye Stains-AII according to the general method, and subsequently analyzed on agarose gel. The HPLC chromatogram and the photograph of the agarose gel are depicted in
Fraction 1 and 2 did not react with the dye, indicating concentrations of SDS and nucleic acids below the detection limit of 21 μmol/L SDS. Fraction 3 and 4 exhibited a blue color upon addition of the dye, indicating the presence of nucleotides or soluble proteins, which cannot be stained with SYBR-Green II on an agarose gel. Fraction 5 and 6 were eluted at conductivities typical for RNA, and gave a faint band of nucleic acid smaller than 100 bp in the agarose gel, while fraction 7 contained the genomic DNA.
In further experiments, samples purified by the method of the present invention were compared to samples purified using the commercially available QIAamp kit by HPLC analysis. To determine the amount of genomic DNA, a calibration curve using increasing amounts of genomic E. coli DNA, purified by anion exchange chromatography, was used, correlating the area under curve (AUC) of the gDNA elution peak with the amount of gDNA loaded onto the column. In
12 samples of 10 mg pork liver were lysed and purified according to the method of the present invention using water as eluent in the gel filtration step, and compared to 6 samples of 10 mg pork liver purified using the QIAamp columns with the same eluent. The results are shown in
To ensure that no PCR inhibitors are present in the samples purified according to the present invention, samples of 20 mg rat tail were lysed and purified according to the method of the present invention as detailed above. These purified samples were then analyzed in a real time PCR (RT-PCR) using the jun-system on the Rotorgene system (Corbett, Sydney, Australia) and compared to the results obtained using gDNA using AEX chromatography. A commercially available primer/probe system (FAM-TAMRA, Applied Biosystems, Darmstadt, Germany), including a 20×jun PCR primer/probe mix was used in combination with an 2×TAQman PCR universal master mix from Applied Biosystems. The RT-PCR was carried out according to the manufacturer's instruction. In particular, polymerase activation was carried out by heating the mixture for 20 min to 95° C., cycling was performed by melting the duplexes at 95° C. for 15 seconds, and annealing was carried out at 60° C. for 60 seconds. In total, 40 cycles were performed. The reaction mixture consisted of 22.5 μL of the gDNA-containing sample, 25 μL of the master mix and 2.5 μL of the primer mix. Samples obtained from the lysis and purification process of the present invention were used in an undiluted form and after dilution with water (factor 10, 100 and 1000) as a template in the PCR. Even though the undiluted sample (curve 1 in
Similar results were obtained with samples derived from 10 mg frozen liver tissue, lysed and purified according to the present invention in comparison to samples lysed and purified using the QIAamp kit.
A jun assay was also performed using samples that were lysed as described above with subsequent SDS removal by precipitation, but were not further purified using the gel filtration step. In this assay undiluted, 2- 4- and 8-fold dilutions were completely inhibited. However, amplification from a 16-fold diluted solution was possible.
Samples of the different tissues listed in Table 2 were lysed as described above and SDS was subsequently precipitated from the lysate by adding strontium ions, or the samples were lysed according the QIAamp protocol. After lysis (and precipitation in the case of the present invention), the samples obtained from the same kind of tissue by the same purification method were pooled, and then split into 100 μL aliquots. These aliquots were purified according to the method of the present invention or according to the QIAamp protocol, respectively. The amount of gDNA present in the sample was analyzed by UV/Vis spectroscopy and/or HPLC analysis. The average results obtained are given in Table 2.
Due to the high liquid content of blood samples, a twofold concentrated lysis buffer (2×) comprising 50 mmol/L TRIS and 50 mmol/L SDS, adjusted to pH 8.5 by the addition of H2SO4, was used for lysis of the cells. Two samples of 40 μL of human blood from the same donor were mixed with an equal amount of the 2× lysis buffer. Blood proteins were then depleted by addition of 10 μL QIAGEN Protease (2.5 AU/ml). The samples were incubated at 62° C. for 10 min. SDS was removed from the lysates by precipitation as described above, and the samples were purified by gel filtration chromatography using the spin columns described above. Aliquots of 5 μL and 10 μL were then analyzed on an agarose gel (
Quantification of the amount of gDNA present within the samples revealed an average yield of 600 ng gDNA in the samples using 18S gDNA primers (for each of the two samples RT-PCR was carried out in duplicate, and the amount of gDNA was determined as an average value from all four experiments).
50 ng (as estimated by UV/Vis spectroscopy) of purified gDNA obtained from rat liver, kidney and spleen tissue was lysed and purified using the method of the present invention, or according the DNeasy protocol using a commercially available DNeasy kit (QIAGEN, Hilden, Germany) were used as targets for RT-PCR. Fresh and frozen tissue samples, and samples stabilized in the commercially available RNAlater reagent (QIAGEN, Hilden, Germany) were used. The samples were then analyzed in a SYBR-Green-based RT-PCR reaction on a TAQman system. As can be seen in
20 mg of rat liver and spleen tissue, respectively, were lysed and purified according to the present invention. Lysis was complete within 30 min. Each experiment was carried out in duplicate. Quality of gDNA in the lysates obtained were analyzed in a PCR reaction and on an ethidium bromide stained agarose gel (
Samples of 10 mg of frozen rat liver tissue each were lysed as described above. After addition of the precipitating agent the samples were purified by gel filtration chromatography, using the columns described above, equilibrated in water and buffer AE, respectively. In addition, several samples were lysed in a twofold concentrated lysis buffer C and purified by gel filtration chromatography using the spin columns of described above, equilibrated in buffer AE. The eluates obtained were analyzed by gel electrophoresis and the amount of gDNA, and SDS as well as the conductivity of the eluates were determined. SDS was only detected in two samples. The results are presented in
Samples of 10 μm thickness from a formalin fixed paraffin embedded (FFPE) block of rat liver were lysed, and the gDNA was isolated and purified using a) a commercially available QIAamp kit (QIAGEN, Hilden, Germany) and b) using the method of the present invention.
a) Three sections with each 10 μm from the FFPE block were lysed in 1 mL xylene, mixed by vortexing for 10 s, and centrifuged at full speed for 2 min. The supernatant was removed by pipetting. Residual xylene was extracted by adding 1 mL EtOH to each sample, vortexing the samples for 10 s, centrifuging them at full speed for 2 min and removing the supernatant. The open tubes containing the samples were incubated at room temperature for 20 min to evaporate residual EtOH. The pellets obtained were resuspended in 180 μL buffer ATL (QIAGEN, Hilden, Germany). To each sample 20 μL of Proteinase K (2.5 AU/ml) (QIAGEN, Hilden, Germany) were added, and the samples were mixed by vortexing. The samples were incubated at 56° C. for 1 h before incubating them at 90° C. for 1 h. The samples were cooled to room temperature, then 1 μL of RNase A (10 U/ml) (QIAGEN, Hilden, Germany) was added to each sample. 200 μL buffer AL (QIAGEN, Hilden, Germany) was added to each lysate, and the samples were mixed thoroughly by vortexing. 200 μL EtOH were then added, and the samples were vortexed again. The lysates were transferred to a QIAamp MinElute column (QIAGEN, Hilden, Germany) and centrifuged at 6000×g for 1 min. The QIAamp MinElute column was placed into a clean 2 mL collection tube and the flow-through was discarded. The column was opened and washed with 500 μL buffer AW1 (QIAGEN, Hilden, Germany) and centrifuged at 6000×g for 1 min. The QIAamp MinElute column was placed into a clean 2 mL collection tube and the flow-through was discarded. The column was opened again and washed with 500 μl buffer AW2 (QIAGEN, Hilden, Germany) and centrifuged at 6000×g for 1 min. The QIAamp MinElute column was placed into a clean 2 mL collection tube and the flow-through was discarded. The membrane was centrifuged at full speed for 3 min to remove traces of buffer. Elution of the gDNA was done in a clean 1.5 ml micro centrifuge tube with 100 μl RNase-free water by incubation at room temperature for 1 min and centrifugation at full speed for 1 min.
b) Three sections with each 10 μm thickness from the FFPE block were lysed in 80 μL of lysis buffer C (25 mmol/l Tris/H2SO4, 25 mmol/L SDS, pH 8.5), supplemented with 10 μL QIAGEN Protease (2.5 AU/ml) (Hilden, Germany) and 1 μL RNase A (7000 U/ml). The samples were mixed by vortexing. Three samples were incubated at 62° C. for 30 min and then at 90° C. for 1 h, while the other three samples were incubated at 56° C. for 1 h before incubating them at 90° C. for 1 h. To each sample 10 μL of the precipitating solution (1 mol/L SrCl2) was added, and the samples were mixed by vortexing and incubated on ice for 10 min. Each sample was then transferred to a pre-spun spin column according to the present invention (“single-step column”), and the column was centrifuged at 700×g for 3 min, while the eluate containing the gDNA was collected.
To evaluate the amount and quality of the gDNA obtained, a SYBR Green-based RT-PCR was carried out on a TAQman system using the gene coding for 18S rRNA as a target. The results are presented in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10003765.4 | Apr 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/055555 | 4/8/2011 | WO | 00 | 10/5/2012 |
