Method for isolating nucleic acid from a biological sample

Abstract

The present invention refers to a method for isolating nucleic acid from a biological sample, comprising: (a) bringing the biological sample into contact with: a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and a C8-C12-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase, (b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about 90°C; (c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and (d) directly transfer of the aqueous phase onto a size exclusion chromatography resin. The present invention further relates to the use of a solution for removing SDS and / or LiDS from a biological sample. Further, the present invention relates to a kit for removing SDS and / or LiDS from a biological sample.

Description

METHOD FOR ISOLATING NUCLEIC ACID FROM A BIOLOGICAL SAMPLEFIELD OF THE PRESENT INVENTION

[0001] The present invention refers to a method for isolating nucleic acid from a biological sample, comprising: (a) bringing the biological sample into contact with: a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and a C8-C12-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase, (b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about 90°C; (c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and (d) directly transfer of the aqueous phase onto a size exclusion chromatography resin. The present invention further relates to the use of a solution comprising a C8-C12-alkane comprising at least one end-terminal hydroxyl-group for removing SDS and / or LiDS from a biological sample. Further, the present invention relates to a kit for removing SDS and / or LiDS from a biological sample.BACKGROUND OF THE PRESENT INVENTION

[0002] Various methods for isolating nucleic acid from biological samples have been known for a long time. Such are, for example, silica-based technologies. In such methods, the nucleic acids, being present for example in a lysate, bind to a silica surface, e.g. a membrane or magnetic beads, while inter alia cell components are washed away by repeated washing with chaotropic and alcohol- containing wash buffers. Eventually, the nucleic acids are eluted with an aqueous buffer in the desired volume. Such a nucleic acid isolation using a silica-based method and in the presence of chaotropic agents is for example described in EP 1932913 Al. However, due to the repeated washing steps, silica-based methods are time-consuming, labor-intensive and environmentally unfriendly. Further, additional steps are usually required to reduce the amount of chaotropic agent for being able to perform subsequent steps, which might be inhibited by such agents.

[0003] Additionally, not only the presence of chaotropic agents, but also of further substances being usually present in lysis buffers can result in disadvantages for performing subsequent steps after nucleic acid isolation, like PCR. Such substances are, for example, detergents, specifically anionic detergents, like SDS or LiDS. Those are known for their protein denaturizing properties. However, thisproperty also leads to the inhibition of, e.g., PCR, even by the presence of very small amounts of SDS. As described above, undesired agents are up to now separated from nucleic acids by binding of the nucleic acids to silica membranes or silica coated magnetic beads. The same applies for detergents like SDS and / or LiDS. Another way known to the person skilled in the art for separating detergents from nucleic acid-containing samples is by precipitation of theses detergents, specifically by precipitation using monovalent ions of alkali metals and / or divalent ions of alkaline earth metals, such as Rb+, Cs+, Ca2+, Sr2+or Ba2+, like described in WO 2011 / 157683 Al. However, this leads to various disadvantages like that completeness of precipitation formation has to be ensured. Additionally, this also requires an additional step being also time-consuming.

[0004] For the separation of various components, EP 2270151 Al describes the formation of different phases like a liquid organic phase and an aqueous phase for isolating biomolecules from a biological specimen. However, this method according to EP 2270151 Al makes use of anionic detergents, which are discarded by the previously known and time-consuming silica based bind- wash-elute methods.

[0005] Thus, different methods for isolating nucleic acids from biological samples are known. Yet, there is still a need for alternative methods. Especially, there is a need for methods that allow rapid nucleic acid isolation from biological samples and in which the amount and quality of the obtained nucleic acids is sufficient for diverse downstream applications, such as nucleic acid amplification methods, like RT-PCT, qPCR, sequencing methods such as Next Generation Sequencing, cloning methods, or the like. The present invention addresses these needs by providing methods, uses and kits that are suitable among other things for a reliable isolation of nucleic acids and suitable for subsequent downstream applications.SUMMARY OF THE PRESENT INVENTION

[0006] The present invention provides a method for isolating nucleic acid from biological samples, such that the application of downstream applications like PCR is not inhibited. The inventors of the present application have surprisingly found that components like SDS and / or LiDS can be sufficiently separated from nucleic acids of a biological sample by bringing the biological sample into contact with a C8-C12-alkane comprising one end-terminal hydroxyl-group and by applying specific, well- defined conditions that allow that a multiple phase mixture is formed.

[0007] According to the method of the present invention, the nucleic acids contained in a biological sample do not have to get bound to a silica membrane or magnetic beads. Instead, a size exclusionchromatography resin is used. Undesired components of a lysate formed from the biological sample by bringing it into contact with a lysis buffer are separated from the nucleic acids contained in the biological sample due to the steps of the method according to the present invention. Therefore, the method according to the present invention does not need time-consuming washing steps and offers easy handling as well as reduced plastic waste.

[0008] The present invention relates to a method for isolating nucleic acid from a biological sample, comprising:(a) bringing the biological sample into contact with:• a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and• a C8-Ci2-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase,(b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about 90°C;(c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and(d) directly transfer of the aqueous phase onto a size exclusion chromatography resin.

[0009] The high yield and purity of the total nucleic acids obtained by the method of the present invention allows the application of several downstream applications, such as NGS, PCR and qPCR, without further processing. The method of the present invention thereby results in short processing time, very few protocol steps, high sample throughput with minor equipment and a high reduction of plastic waste as well as the avoidance to use toxic reagents.

[0010] The method of the present invention comprises lysis and a single-step purification. The purification step works differently than conventional methods, such as magnetic beads and silica kits based on the bind-wash-elute method and therefore needs only a reduced number of centrifugation steps, preferably just one centrifugation step.

[0011] In the method of the present invention, the C8-C12-alkane comprising at least one endterminal hydroxyl-group is preferably selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, more preferably the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1- decanol.

[0012] The present invention can be performed without the necessity to use any chaotropic agent(s), thereby avoiding the disadvantages arising from the presence of such agents. Therefore, inone embodiment of the method of the present invention, step (a) is conducted in absence of (a) chaotropic agent(s). In one further embodiment of the method of the present invention, the lysis buffer does not comprise (a) chaotropic agent(s).

[0013] The present invention can be performed without the necessity to apply a precipitation step, thereby avoiding the disadvantages arising from such a step. It could be performed, however, it is not necessary, e.g. for removing components like SDS and / or LiDS, because, with the method of the present invention, the skilled person is able by other means described herein in detail to remove those detergents in a sufficient way for being able to perform downstream applications afterwards. Therefore, in one preferred embodiment of the method of the present invention, the method is without a SDS and / or LiDS precipitation step. It is also preferred for the method of the present invention that it is without the presence of monovalent ions of alkali metals and / or divalent ions of alkaline earth metals, more preferably selected from the group consisting of Rb+, Cs+, Ca2+, Sr2+and Ba2+in an amount and / or quality that would be sufficient to precipitate SDS and / or LiDS comprised in the buffers, in particular the lysis buffer.

[0014] In one embodiment of the method of the present invention, step (b) is carried out for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, preferably for at least 5 minutes.

[0015] In one embodiment of the method of the present invention, the biological sample is a wax- embedded biological sample. In one preferred embodiment, the wax-embedded biological sample is a formalin-fixed paraffin-embedded (FFPE) biological sample, more preferably a FFPE feces sample, a FFPE cell culture sample, a FFPE blood sample, a FFPE urine sample, a FFPE tissue sample or a FFPE body fluid sample.

[0016] In one further embodiment of the method of the present invention, bringing the biological sample into contact with the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is after the paraffin of the FFPE biological sample is dissolved or has been dissolved.

[0017] In one embodiment of the method of the present invention, the method further comprises (an) enzymatic digestion step(s) at a temperature of at least 60°C, preferably comprising mixing, preferably for about 30 minutes. It is preferred that at least one enzyme(s) is / are used for the enzymatic digestion step(s). It is further preferred that with the enzymatic digestion step(s), components other than the nucleic acids are enzymatically digested. It is further preferred that theat least one enzyme(s) is / are lytic enzyme(s). It is even more preferred that the at least one enzyme(s) is / are selected from the group consisting of proteases, lipases, cellulases, hydrolases, chitinases, amylases and glucanases. It is further preferred that hydrolases are not nucleases.

[0018] In one further embodiment of the method of the present invention, the at least one enzyme(s) comprise(s) at least one protease(s). Preferably, the at least one protease(s) is / are selected from the group consisting of protease from Bacillus licheniformis, protease from Bacillus spec., protease from Staphylococcus aureus, protease from Bacillus amyloliquefaciens, protease from Coprinus spec, and protease from Aspergillus oryzae.

[0019] In one embodiment of the method of the present invention, said size exclusion chromatography resin of step (d) is a size exclusion chromatography resin with exclusion limits in the range of 20 to 2000 bp of single and / or double stranded nucleotide strands. Preferably, said size exclusion chromatography resin is incorporated into a spin column.

[0020] The present invention further relates to the use of a solution comprising a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1- dodecanol or mixtures thereof, more preferably wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, for removing SDS and / or LiDS from a biological sample.

[0021] In one further aspect, the present invention relates to a kit for removing SDS and / or LiDS from a biological sample, comprising(i) a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8- C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1- decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, and(ii) at least one component(s) selected from the group consisting of a lysis buffer, an agent for enzymatic digestion, preferably a protease, RNase or DNAse, a buffer, MgCI2solution and a size exclusion chromatography resin.BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 shows a schematic representation of an FFPE block and an FFPE section. Grey rectangle: areas with paraffin only; white area: area with tissue fixed in formalin and embedded in paraffin; black rectangle: area to be used for FFPE tissue extraction.

[0023] Figure 2 shows the principle of the single-step-purification used in the method of the present invention.

[0024] Figure 3 shows the standard diagram / curve for Example 2.

[0025] Figure 4 shows the results obtained in Example 3.

[0026] Figure 5 shows the standard diagram / curve for Example 3.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0027] The present invention relates to a method for isolating nucleic acid from a biological sample, comprising:(a) bringing the biological sample into contact with:• a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and• a C8-Ci2-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase,(b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about 90°C;(c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and(d) directly transfer of the aqueous phase onto a size exclusion chromatography resin.

[0028] The term "isolating" or "isolation" (or the like) of nucleic acids as used herein may mean the purification of nucleic acids. For example, isolating or the isolation may mean that the nucleic acids are extracted from a sample, such as a biological sample. It is envisioned that the extraction of the nucleic acids from the sample can include that the nucleic acids are brought into solution.

[0029] Nucleic acids as described herein may refer to any nucleic acid. For example, the nucleic acid may be RNA or DNA, preferably DNA. RNA as used herein may refer to any RNA. For example, the RNA may be mRNA, tRNA or rRNA. For example, the DNA may be genomic DNA, circulating DNA or plasmid DNA.

[0030] The method of the present invention relates to the isolation of nucleic acids from a biological sample. A "biological sample" as used herein, refers to any biological material containing nucleic acids. In one embodiment, biological samples as used herein may comprise cells and / or cell-free nucleic acids from gram-positive or gram-negative bacteria, virus, protozoa, chromista, fungi, plants and / or animals. In another embodiment, the biological sample is isolated from fungi, plants and / or animals, but may contain a biological sample consisting of cells from bacteria, protozoa, chromista, fungi, plants and / or animals. In one embodiment, animal refers to vertebrates, preferably tetrapods, fish, and / or birds, more preferably mammals and even more preferably cows, cats, dogs, horses, pigs or humans. It is also envisioned that the biological sample is a blood sample, preferably a human blood sample, or a tissue sample, preferably a muscle sample, sperm sample, plant sample, cell sample, mucosa sample, such as an oral mucosa sample or a bacterial sample.

[0031] According to the method of the present invention, the biological sample is contacted with a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and a C8-Ci2-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase.

[0032] Within the context of the present invention, a lysis buffer is understood as being a solvent comprising at least one compound(s), which is / are capable of releasing nucleic acid from a biological sample or material of a biological specimen. These at least one compound(s) can be at least one enzyme(s), for example, protease(s). Other substances supporting the lysis can also be used within the context of the present invention.

[0033] The term "liquid organic phase" as used herein and in the context of the present invention means a phase, comprising a C8-Ci2-alkane comprising at least one end-terminal hydroxyl -group, preferably 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, more preferably 1-decanol, characterized by minimal solubility with water. According to the present invention, the liquid organic phase is a solvent, which is less than 5 %, preferably less than 2 %, more preferably less than 1 % and even more preferably less than 0.5 % soluble in water. The preferred 1-decanol is in the context of the present invention not soluble in water at a temperature of 25°C. The method according to the present invention makes use of an organic phase that is liquid at normal ambient temperatures, in particular by 25°C.

[0034] The term "aqueous phase" as used herein and in the context of the present invention means a water-based phase, which is characterized by minimal solubility with an organic phase. According to the present invention, the aqueous phase is a solvent, which is less than 5 %, preferably less than2 %, more preferably less than 1 % and even more preferably less than 0.5 % soluble in an organic phase.

[0035] The term "multiple phase mixture" as used herein and in the context of the present invention means that the phases form distinct regions in said mixture with different physical and / or chemical properties. The multiple phase mixture according to the present invention comprises at least two phases, namely the two phases of step (a), the aqueous phase and the liquid organic phase. In one embodiment, the multiple phase mixture comprises the aqueous phase of step (a), the liquid organic phase of step (a) and a further / third phase, which appears between the aqueous phase and the liquid organic phase. Such a further / third phase is preferably formed, when the biological sample is a FFPE biological sample. Thus, in one embodiment of the present invention, the multiple phase mixture comprises three phases. In one embodiment, the aqueous phase of the multiple phase mixture comprises the nucleic acids.

[0036] According to the present invention, the liquid organic phase of the multiple phase mixture comprising the C8-Ci2-alkane comprising one end-terminal hydroxyl-group may, after steps (b) and (c), further comprise the SDS and / or LiDS. This means that steps (b) and (c) of the present invention enable the transition of the SDS and / or LiDS out of the aqueous phase, preferably into the liquid organic phase. Besides the aqueous phase and that liquid organic phase, a further phase may be present. Such a further phase may be also called "third phase" or "interphase" in the context of the present invention. Said third phase is between the aqueous phase and the liquid organic phase. It is preferred that the aqueous phase is the phase at the bottom of the multiple phase mixture. It is also preferred that the liquid organic phase is the phase at the top of the multiple phase mixture.

[0037] It is preferred that the interphase is characterised by being solid at room temperature. This means that preferably with the interphase a solid state phase is formed. The terms "solid" and "solid state" are known to the person skilled in the art. It is preferred for said interphase that it is insoluble at room temperature in both phases, the aqueous phase and the liquid organic phase. The term "insoluble" means in the context of the present invention that the interphase is less than 5 %, preferably less than 2 %, more preferably less than 1 % and even more preferably less than 0.5 % soluble in the other phases, the aqueous phase and the liquid organic phase. The interphase is preferably formed after mixing and / or centrifugating in step (c) of the method of the present invention. Said interphase is between the liquid organic phase and the aqueous phase, preferably with the aqueous phase being below and the liquid organic phase being above the interphase. It is preferred for the interphase that it essentially comprises wax and / or paraffin of the biologicalsample. The term "essentially" means here that at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the interphase comprises or consists of wax and / or paraffin.

[0038] The term "directly" as used in step (d) of the method of the present invention, means in the context of the present invention that as soon as the multiple phase mixture is formed in step (c) of the method of the present invention, the aqueous phase of the multiple phase mixture is transferred without hesitation onto the size exclusion chromatography resin and that size exclusion chromatography is performed after that transfer.

[0039] It is also envisioned by the inventors of the present invention that the biological sample may be contacted with a lysis buffer comprising any anionic detergent to form an aqueous phase in step (a) of the method of the present invention. Non-limiting examples of anionic detergents include lithium dodecyl sulfate (LiDS), NaDS, sodium octyl sulfate, decyltrimethylammonium chloride, potassium oleate, sodium pentanesulfonate, sodium dodecyl sulfate, butylnaphthalensulfonic acid sodium salt, 4-morpholineethanesulfonic acid, sodium decyl sulfate, lignosulfonic acid calcium salt, sodium 1-butanesulfonate, sodium dodecylbenzenesulphonate, sodium stearate, magnesium stearate, 1-dodecanesulfonic acid sodium salt, sodium allylsulfonate, 3-(N,N- dimethylpalmitylammonio)propanesulfonate, sulfonated castor oil, 2,6-dimorpholin-4-ylpyrimidine- 4-carboxylic acid, disodium methylenebisnaphthalenesulphonate, sodium alkylbenzene sulfonate, hydroxyaluminum distearate, sodium ethyl 2-sulfolaurate, sodium diisobutyl sulfosuccinate, dodecylbentenesulfonic acid sodium salt, dicyclohexyl sulfosuccinate sodium salt, disodium 4- dodecyl-2,4'-oxydibenzenesulfonate, sulfonated aliphatic polyester, sodium-n-methyl-n-oleyl taurate, di-n-sodium sulfosuccinate, dibasic lead stearate, sodium n-octylsulfonate, dodecyl triethanolamine sulfate, sodium diamyl sulfosuccinate, manganous stearate, calcium dodecylbenzene sulfonate, disodium 4-[2-[(l-2-sulphonatosuccinate-poly[(naphthaleneform- aldehyde)sulfonate], 1-hexadecanesulfonic acid sodium salt, ammonium lauryl sulfate, 1- pentanesulfonic acid sodium salt monohydrate, sodium lignosulfonate, dodecylbenzenesulphonic acid, sodium lauryl polyoxyethylene ether sulfate, sodium nonylphenol polyoxyethylene ether sulfate, sodium dodecyl sulfate, fatty alcohol ammonium sulfate, sodium oleyl sarcosinate, lauryl polyoxyethylene ether triethanol amine salt, dodecyl phenyl ammonium sulfate, sodium pyrrolidone carbonate, n-acyl glutamate potassium salt, sodium polyalkyl phenyl polyoxyethylene ether sulfate, stearyltoluene sodium sulfonate, nonylphenyl polyoxyethylene ether sulfate triethanolamine, glyceryl ether carboxylic acid salt, calcium stearyl lactate, monoethanolamine dodecyl sulfate, alkoxy ethanolamido sulfosuccinate sodium salt, ammonium dodecylbenzenesulphonate, dodecay diethanolamine sulfate, sodium dibenzyl amine benzene sulfonate or sodium dibenzyl amine benzene sulfonate.

[0040] The amount and quality of the nucleic acids as obtained by the method according to the present invention is sufficient so that these nucleic acids can be used in diverse downstream applications. Thus, the isolated nucleic acids / eluate (including the isolated nucleic acids in solution) can be analyzed by all conventional molecular techniques, such as, for example, PCR, next generation sequencing, SNP genotyping, qPCR or RT-PCR. The isolated nucleic acids as received after step (d) of the method of the present invention can therefore be analyzed by methods, such as PCR next generation sequencing, SNP genotyping or RT-PCR without the need of further isolating steps. The nucleic acids of the biological sample may be ribonucleic acids (RNA) or desoxyribonucleic acids (DNA), preferably DNA, in the context of the present invention.

[0041] According to the method of the present invention, in step (a) the biological sample is brought into contact with a C8-C12-alkane comprising at least one end-terminal hydroxyl-group. With the addition thereof to the biological sample, a liquid organic phase is formed. In one embodiment of the method of the present invention, the C8-C12-alkane comprising at least one end-terminal hydroxylgroup is a C8-Ci2-alcohol.

[0042] In one preferred embodiment of the method of the present invention, the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is a C8-Ci2-alkane comprising at least one endterminal hydroxyl-group immiscible with water. Such a C8-C12-alkane comprising at least one endterminal hydroxyl-group immiscible with water is understood as being a respective solvent, which at 25 °C., with a mix ratio of 1:1 with water, forms two phases separated from one another. In one embodiment of the method according to the present invention, such a solvent immiscible with water has a lesser density than water and / or the aqueous phase. The density of such a solvent immiscible with water may be in particular < 0.95 g / cm3, preferably < 0.90 g / cm3. That solvent immiscible with water is preferably less than 5 %, more preferably less than 2 %, even more preferably less than 1 % and even more preferably less than 0.5 % soluble in water. In a further embodiment of the method according to the present invention, the solvent immiscible with water is liquid at < 25 °C, preferably is liquid at a temperature < 20 °C, more preferably is liquid at a temperature of < 15 °C, even more preferably is liquid at a temperature < 10 °C.

[0043] In one preferred embodiment of the method of the present invention, the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof. More preferably, the C8-Ci2-alkane comprising at least one endterminal hydroxyl-group is 1-decanol.

[0044] In one preferred embodiment, the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is 1-octanol. 1-Octanol has the following chemical structurethe molecular formula CH3(CH2)7OH.

[0045] In one further preferred embodiment, the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol. 1-Decanol has the following chemical structurethe molecular formula C10H21OH.

[0046] In one further preferred embodiment, the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-dodecanol. 1-Dodecanol has the following chemical structureand the molecular formula C12H26O.

[0047] It is especially beneficiary that 1-decanol is used as the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group. 1-Decanol is especially beneficiary for enabling a transition of SDS and / or LiDS out of the aqueous phase of step (a), preferably into the liquid organic phase. This also applies for embodiments, wherein the multiple phase mixture comprises three phases, e.g. an interphase, which is present in addition to the aqueous phase and the liquid organic phase. Thus, in one embodiment of the method of the present invention, after steps (b) and (c) of the method of the present invention, the aqueous phase is essentially free from SDS and / or LiDS. Preferably, the SDS and / or LiDS have / has essentially transitioned out of the aqueous phase, more preferably into the other phase or the other phases of the multiple phase mixture. Even more preferably, SDS and / or LiDS has / have essentially transitioned into the liquid organic phase of the multiple phase mixture.

[0048] In one preferred embodiment of the method of the present invention, step (a) is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes, preferably for at least 40 minutes.

[0049] In one preferred embodiment, SDS and / or LiDS is present in step (a) in a concentration of at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, atleast 130 mM, at least 140 mM, at least 150 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 M or at least 200 mM.

[0050] In one further preferred embodiment of the method of the present invention, SDS and / or LiDS is present in step (a) at a concentration in the range of about 10 mM to about 200 mM, preferably at a concentration in the range of about 20 mM to about 150 mM, more preferably at a concentration in the range of about 50 mM to about 100 mM.

[0051] In addition to the lysis buffer, further substances can be present in the aqueous phase, such as, for example, at least one buffer substance. Appropriate buffer substances are, for example, Tris / HCI, phosphate buffer, borate buffer, PBS buffer, citrate buffer, MES buffer or HEPES buffer.

[0052] In one embodiment of the method of the present invention, step (a) is conducted in absence of (a) chaotropic agent(s). This means, that within the context of the present invention, the presence of (a) chaotropic agent(s) may not be necessary for achieving a sufficient lysis with the applied lysis buffer. Thus, in one embodiment of the method of the present invention, the lysis buffer as used in step (a) does not comprise (a) chaotropic agent(s). A "chaotropic agent" is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules (i.e. exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids and may cause its denaturation. Chaotropic agents might be, but are not limited to, NaBr, Nal, NaSCN, LiCI, LiBr, NH4AC, NaCI, guanidinium chloride, guanidinium hydrochloride, lithium perchlorate, sodium perchlorate, lithium acetate, magnesium chloride, guanidinium isothiocyanate or guanidinum isocyanate (GuSCN).

[0053] In one preferred embodiment of the method of the present invention, the method is without a SDS and / or LiDS precipitation step. It could be performed, however, it is not necessary, e.g. for removing components like SDS and / or LiDS, because, with the method of the present invention, the skilled person is able by other means described herein to remove those detergent(s) in a sufficient way for being able to perform downstream applications afterwards. This means that the method according to the present invention does not require a precipitation step for separating the SDS and / or LiDS from the nucleic acid of the biological sample. This may also mean that in one embodiment, the method is conducted in absence of monovalent ions of alkali metals and / ordivalent ions of alkaline earth metals, preferably selected from the group consisting of Rb+, Cs+, Ca2+, Sr2+and Ba2+in an amount and / or quality that would be sufficient to precipitate SDS and / or LiDS comprised in the buffers, in particular the lysis buffer. It is further preferred that in the context of the present invention, the method is conducted in the absence of a clearing solution, for example, for precipitating the anionic detergents SDS and / or LiDS. Such a clearing solution may usually comprise K+, Rb+, Cs+, Mg2+, Ca2+, Sr2+or Ba2+in an amount and / or quality that would be sufficient to precipitate SDS and / or LiDS comprised in the buffers, in particular the lysis buffer.

[0054] In a preferred embodiment of the method of the present invention, the method is conducted / carried out without a bind-wash-elute-step. This allows to safe a tedious step being one of the most time-consuming steps.

[0055] In one embodiment of the method of the present invention, it is preferred that step (b) is carried out for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, preferably for at least 5 minutes. It is preferred for step (b) that mixing of the two phases of step (a) is at a temperature in the range of about 85°C to about 90°C. It is further preferred for step (b) of the method of the present invention that mixing the two phases of step (a) is at a temperature of about 90°C. In the context of the present invention and as used herein, the term "about" followed by a temperature value means the temperature value as such + / - 5°C, preferably + / - 2°C and more preferably + / - 1°C of that temperature value.

[0056] It is within the scope of the present invention that the biological sample may be a wax- embedded biological sample. Preferably, the wax-embedded biological sample is a formalin-fixed paraffin-embedded (FFPE) biological sample. It is more preferred that the FFPE biological sample is a FFPE feces sample, a FFPE cell culture sample, a FFPE blood sample, a FFPE urine sample, a FFPE tissue sample or a FFPE body fluid sample.

[0057] In preferred embodiments, the biological sample is a body fluid sample, a stool sample, an environmental sample, a cell culture sample, a bone marrow sample, a sewage sample, a food sample, a milk sample, a forensic sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, and any combination thereof, wherein, optionally, the body fluid sample is one of a blood sample, a serum sample, an amniotic fluid sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swabsample, a nasal swab sample, a bronchoalveolar lavage sample, a bronchial secretion sample, and an urine sample.

[0058] In one embodiment of the method of the present invention, bringing the biological sample into contact with the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is after the paraffin of the FFPE biological sample is dissolved or has been dissolved. This means, if the biological sample is a FFPE biological sample, which comprises wax and / or paraffin, then the wax and / or paraffin has been essentially dissolved before the biological sample is brought into contact with the C8-C12-alkane comprising at least one end-terminal hydroxyl-group. "Essentially dissolved" means in this regard that less than 2 % of the total amount of wax / paraffin of the FFPE biological sample has not been dissolved, preferably that less than 1 % of the total amount of wax / paraffin of the FFPE biological sample has not been dissolved, more preferably that less than 0.5 % of the total amount of wax / paraffin of the FFPE biological sample has not been dissolved and even more preferably that less than 0.1 % of the total amount of wax / paraffin of the FFPE biological sample has not been dissolved. Thus, the wax and / or paraffin of the FFPE biological sample is dissolved by other means than by bringing the FFPE biological sample into contact with the C8-Ci2-alkane comprising at least one endterminal hydroxyl-group. The person skilled in the art knows how to dissolve the wax and / or paraffin of such a FFPE sample and is well aware of the methods or means, which enable the dissolution thereof.

[0059] The multiple phase mixture according to the present invention comprises at least two phases, namely the two phases of step (a), the aqueous phase and the liquid organic phase. In one embodiment, the multiple phase mixture comprises the aqueous phase of step (a), the liquid organic phase of step (a) and a further / third phase. That further / third phase is described herein above. Thus, in one embodiment of the present invention, the multiple phase mixture comprises three phases. In one embodiment, the aqueous phase of the multiple phase mixture comprises the nucleic acids.

[0060] Also for these embodiments, wherein a FFPE biological sample is applied to the method of the present invention as biological sample, the liquid organic phase of the multiple phase mixture comprising the C8-Ci2-alkane comprising one end-terminal hydroxyl-group may, after steps (b) and (c), additionally comprise the SDS and / or LiDS. This means that steps (b) and (c) of the present invention enable the transition of the SDS and / or LiDS out of the aqueous phase, preferably into the liquid organic phase, of the multiple phase mixture also when applying a FFPE biological sample as biological sample. Besides the aqueous phase and the liquid organic phase, a further phase may bepresent. That further phase may be also called "third phase" or "interphase" in the context of the present invention. Said third phase may be between the aqueous phase and the liquid organic phase of the multiple phase mixture. That interphase / third phase is described herein above. Said third phase may be characterized by comprising the wax and / or paraffin of the FFPE biological sample. It is preferred for these embodiments that the aqueous phase is the phase at the bottom of the multiple phase mixture. It is also preferred for these embodiments that the liquid organic phase is the phase at the top of the multiple phase mixture. The third phase, also called interphase herein, is formed as a phase between the liquid organic phase and the aqueous phase of the multiple phase mixture.

[0061] In one embodiment of the method of the present invention, the method further comprises (an) enzymatic digestion step(s) at a temperature of at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 66°C, at least 67°C, at least 68°C, at least 69°C, at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, at least 75°C, at least 76°C, at least 77°C, at least 78°C or at least 80°C. It is preferred that the enzymatic digestion step(s) is / are for digesting non-nucleic acid components comprised in the biological sample.

[0062] As used herein, the term "non-nucleic acid components" comprises all non-nucleic acid compounds in a solution, especially those that compromise or even inhibit subsequent or downstream applications like PCR, cloning, ligation and / or sequencing of nucleic acids. Especially comprised by the term "non-nucleic acid components" are proteins, salts, chaotropic agents, detergents, organic or inorganic solvents, dyes, metabolites, sample debris, low molecular molecules (e.g. nucleotides etc.) and / or PCR inhibitors.

[0063] It is preferred for this embodiment that the enzymatic digestion step(s) comprise(s) mixing. It is also preferred for this embodiment that the enzymatic digestion step(s) is / are carried out for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, more preferably for about 30 minutes.

[0064] Further, it is preferred that at least one enzyme(s) is / are used for the enzymatic digestion step(s). The mentioned "enzyme" as used herein and in the context of the present invention includes enzymes that are typically used in nucleic acid isolation methods, such as proteases, lysozymes, lipases, cellulases, hydrolases, chitinases, amylases or glucanases. More preferably, the at least one enzyme(s) is / are lytic enzyme(s). It is also preferred for this embodiment that the at least oneenzyme(s) is / are selected from the group consisting of proteases, lipases, cellulases, hydrolases, chitinases, amylases and glucanases. It is preferred that hydrolases are not nucleases.

[0065] In one further embodiment of the method of the present invention, the at least one enzyme(s) comprise(s) at least one protease(s). Exemplary proteases include subtilisins, subtilases and alkaline serine proteases. Exemplary subtilisins include proteinase K, proteinase R, proteinase T, subtilisin A, subtilisin B or thermitase. It is preferred that the at least one protease(s) is / are selected from the group consisting of protease from Bacillus licheniformis, protease from Bacillus spec., protease from Staphylococcus aureus, protease from Bacillus amyloliquefaciens, protease from Coprinus spec, and protease from Aspergillus oryzae.

[0066] Step (c) of the method of the present invention is to allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a). This means that according to the method of the present invention, the multiple phase mixture of step (c) comprises at least the aqueous phase and the liquid organic phase of step (a). In one preferred embodiment of the method of the present invention, to allow the mixture of step (b) to form a multiple phase mixture is carried out by centrifugation of the mixture of step (b), preferably at at least 400 g, at at least 500 g, at at least 600 g, at at least 700 g, at at least 800 g, at at least 900 g, at at least 1000 g, at at least 1100 g, at at least 1200 g, at at least 1300 g, at at least 1400 g, at at least 1500 g, at at least 1600 g, at at least 1700 g, at at least 1800 g, at at least 1900 g, at at least 2000 g, at at least 3000 g, at at least 4000 g, at at least 5000 g, at at least 6000 g, at at least 7000 g, at at least 8000 g, at at least 9000 g, at at least 10000 g, at at least 11000 g, at at least 12000 g, at at least 13000 g, at at least 14000 g, at at least 15000 g, at at least 16000 g, at at least 17000 g, at at least 18000 g, at at least 19000 g or at at least 20000 g. In another preferred embodiment of the method of the present invention, to allow the mixture of step (b) to form a multiple phase mixture is carried out by allowing the mixture of step (b) to stand until phase separation occurs, preferably for at least 5 minutes, at least 10 minutes, at least 25 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes.

[0067] In step (d) of the method of the present invention, a size exclusion chromatography (SEC) resin is used. The aqueous phase being comprised in the multiple phase mixture is directly transferred onto said resin after step (c). As used herein, the term "resin" comprises an insoluble matrix or medium capable of interacting with binding partners. A resin is used in a chromatographic procedure like SEC, wherein the resin retains different components depending on theircharacteristics to a different extent and thereby separates the different components of the solution or mixture.

[0068] In one preferred embodiment, step (d) may further comprise (an) enzymatic digestion step(s) of the aqueous phase of the multiple phase mixture. For such an enzymatic digestion step or enzymatic digestion steps, the aqueous phase of the multiple phase mixture may be separated from the other phases of the multiple phase mixture. Such an enzymatic digestion step or enzymatic digestion steps is / are preferably carried out at room temperature. Such an enzymatic digestion step or enzymatic digestion steps may be (an) RNase digestion step(s) and may be performed, when DNA is to be isolated from the biological sample. That enzymatic digestion step or enzymatic digestion steps may be (an) DNase digestion step(s) and may be performed, when RNA is to be isolated from the biological sample. It is preferred for that embodiment that such an enzymatic digestion step or enzymatic digestion steps comprise(s) mixing. Such an enzymatic digestion step or steps may be carried out at room temperature or at a temperature in the range of about 35°C to about 40°C, preferably at about 37°C. That enzymatic digestion step or steps may be carried out for at least 5 minutes, at least 10 minutes, at least 15 minutes, optionally followed by an additional incubation step at about 60°C to about 70°C, preferably at about 65°C.

[0069] Being widely used for the isolation and separation of nucleic acids and nucleic acid mixtures, size exclusion chromatography is known to the person skilled in the art. Size exclusion chromatography filter materials are also known to the person skilled in the art. The principle of SEC is based on the separation of molecules according to their size. It is possible to separate very large and very small molecules from each other (group separation) or several large molecules that only differ from each other in molecular size (high-resolution fractionation). Usually, chromatography columns are filled with the filter material and the analyte mixture to be separated is processed through the column with a mobile phase. The main factors influencing the retention time of the analytes in the column are the particle and pore size of the filter materials used and the flow rate of the mobile phase through the column. Normally, the exact pore size of the filter materials is not given, instead a size exclusion limit in the unit Dalton [Da] is provided. Especially for the separation of large biomolecules (e.g. proteins or nucleic acids) from small molecules (e.g. salts, metabolites, dyes), the manufacturers of SEC filter materials recommend materials with the smallest possible pore sizes (e.g. Cytiva Sephadex resins). The separation of large biomolecules (usually mixtures of proteins) from each other, on the other hand, is more likely to be achieved by using large-pored filter materials (e.g. Cytiva Superdex, Superose and Sephacryl resins).

[0070] Thus, as used herein, the term "size-exclusion chromatography", also known as molecular sieve chromatography, means any chromatographic method, in which molecules in solution are separated by their size, and in some cases by their molecular weight. As used herein, "positive chromatography" refers to a method of enriching a compound by retaining the compound to be enriched in a chromatography device, whereas undesired contaminants, inhibitors and other components are washed away and the compound to be enriched is eluted in a final step. "Negative chromatography" herein refers to a method of enriching a compound by retaining the undesired contaminants in a chromatography device and / or a resin, while the compound to be enriched passes the chromatography device.

[0071] In SEC, the size exclusion limit defines the molecular weight or length of a nucleic acid, where molecules are too large to be trapped in the stationary phase / the resin. The size-exclusion limit of a resin is defined by the composition of the resin and can be influenced by particle size, the type of resin and the degree of crosslinking.

[0072] In one embodiment of the method of the present invention, the size exclusion limit of the SEC resin is between 1 and 10sbase pairs (bp). In a preferred embodiment, the size exclusion limit is between 5 and 10000 bp. In a more preferred embodiment, the size exclusion limit is in the range of 20 to 2000 bp. As used herein, the units "base pairs" (bp) and "nucleotides" (nt) can be used interchangeably.

[0073] In one embodiment of the method of the present invention, the size exclusion chromatography resin of step (d) is a size exclusion chromatography resin with exclusion limits in the range of 20 to 2000 bp of single and / or double stranded nucleotide strands, preferably with exclusion limits in the range of 50 to 2000 bp of single and / or double stranded nucleotide strands, more preferably with exclusion limits in the range of 100 to 2000 bp of single and / or double stranded nucleotide strands, more preferably with exclusion limits in the range of 200 to 2000 bp of single and / or double stranded nucleotide strands, more preferably with exclusion limits in the range of 500 to 2000 bp of single and / or double stranded nucleotide strands, even more preferably with exclusion limits in the range of 1000 to 2000 bp of single and / or double stranded nucleotide strands. Preferably, said size exclusion chromatography resin is incorporated into a spin column.

[0074] Preferably, a resin for size exclusion chromatography may be a hydroxylated methacrylic polymer or a cross-linked dextrane, preferably a dextrane cross-linked with N,N'-methylene- bisacrylamide. Preferably, such a resin for size-exclusion chromatography may also have a water-based mobile phase, such as water, an aqueous organic solvent or an aqueous buffer / solution mobile phase. Commonly, a solid matrix is able to form a gel bed, when suspended in an aqueous medium. Components of such a solid matrix may comprise Sephadex, Sephacryl, hydroxylated methacrylic polymers, crosslinked agarose, silica-based materials, diatomaceous earth, polystyrene / divinyl benzene and / or ceramic hydroxy apatite. One or more components may also be mixed. The one or more component may be suspended in a buffer and may be packed in the hollow body of a column. Columns may be made of glass, plastic, teflon or any other material that neither reacts with the mobile phase nor the analyte. The bead or amorphous particle size of such a medium or resin (material) can range from 1 pm to 500 pm, preferably from 25 pm to 400 pm. The average diameters of such a material or resin may depend on volume and the debris concentration loaded onto the respective resin or column beds.

[0075] In a more preferred embodiment of the method of the present invention, the medium of the size-exclusion chromatography is a resin selected from the group consisting of Sephacryls, preferably Sephacryl 100, Sephacryl 200, Sephacryl 300, Sephacryl 400 or Sephacryl 500, more preferably Sephacryl 400; Toyopearls, preferably Toyopearl HW 65 S, Toyopearl HW 65 F or Toyopearl HW 65 C, and a SEC resin comprising cross-linked agarose, like WorkBeads from Bioworks, e.g. WorkBeads 40 / 10000, 40 / 1000 and 40 / 100.

[0076] The aqueous phase of the multiple phase mixture to be purified may be applied to the gel's bed upper surface of the SEC resin and allowed to pass through the gel, e.g. forced by centrifugation, vacuum or pressure. The aqueous phase of the multiple phase mixture may be the bottom phase of the multiple phase mixture of step (c). The aqueous phase of the multiple phase mixture may, for example, be gained from the multiple phase mixture by sucking away or sucking on the bottom phase from the multiple phase mixture with the help of a pipette or similar device.

[0077] Within this invention, preferably centrifugal forces are applied to move the mobile phase used in the SEC down the column, wherein the columns are spun in a centrifuge (so-called spin column technique, "centrifugation column"). Due to the nature of the resin, pores of a certain size exist inside the gel. Small molecules are able to penetrate the pores, and therefore move through the resin 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 nucleic acid, is then collected at the outlet of the column. To retain the resin 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, wherein nucleic acids of all sizes may pass said frit, filter, fleece or membrane.

[0078] The resin is preferably incorporated into a column. This column may comprise a hollow body having an inlet and an outlet. The hollow body may comprise a solid matrix providing size excluding properties. Preferably, the column may additionally comprise a porous frit, filter, fleece or membrane, preferably allowing nucleic acids of any size to pass, placed between the outlet and the resin. The column may optionally comprise a non-porous ring placed between the porous frit, filter, fleece or membrane and the resin, sealing the outer area of the frit, filter, fleece or membrane, to prevent the mobile phase from entering the frit without passing the resin. Also, optionally, the column comprises at least one removable closing device to seal the inlet and / or the outlet of the chromatographic unit. Further, optionally, the column comprises at least one collection tube to collect the mobile phase (eluate) after having passed the resin. The material of the column may be selected from the group consisting of glass, polypropylene, polycarbonate or polyethylene.

[0079] Preferably, in one embodiment of the method of the present invention, well plates are used for step (a) and / or (b) and / or step (c) of the method of the present invention. Especially, well plates or microplates of the type 96-well- or 384-well-microtiter-plates are used. For example, polystyrene multititer-plates for immunoassay and high throughput screening applications may be used.

[0080] It is preferred that after step (d) of the method of the present invention, an eluate is obtained containing the nucleic acid being essentially free from SDS and / or Li DS. The term "eluate", as used in the context of the present invention, is the product of applying the provided aqueous phase of the multiple phase mixture to step (d) of the method of the present invention as defined herein. Step (d) results in purifying the nucleic acid from the aqueous phase of the multiple phase mixture with size-exclusion chromatography. Thus, the aqueous phase of the multiple phase mixture is transferred onto or is contacted with a medium for size-exclusion chromatography and the sizeexclusion chromatography is performed. The product of this procedure is the so called eluate, which may be collected in the method of the present invention. Thus, a negative chromatography as defined above is applied. The term "contacting" may mean to bring the aqueous phase of the multiple phase mixture into any form of contact with the medium for size-exclusion chromatography, for example, in a column for a certain time or time range. The eluate may then be, for example, subjected to further steps, e.g. for increasing the concentration of the gained nucleic acid in the eluate, before it may be directly applied to PCR, RT-PCR or NGS afterwards.

[0081] In a preferred embodiment of the method of the present invention, the nucleic acid obtained after step (d) is directly applied to PCR, RT-PCR or NGS. For the PCR, the RT-PCR or the NGS, it is preferred that the polymerase used for PCR, RT-PCR or NGS is selected from the group consisting of Taq-polymerase, 7jfl-polymerase, Tmo-polymerase, Tne-polymerase, Tth-polymerase, Pfu- polymerase, Pwo-polymerase, / COD-polymerase, 77 / -polymerase, Tog-polymerase, Tce-polymerase, Tgo-polymerase, T / VAl-polymerase, Tpe-polymerase, Tth / '-polymerase, / Veg-polymerase, Pab- polymerase, T4-DNA-polymerase, T6-DNA-polymerase and T7-DNA-polymerase. More preferably, the polymerase used for PCR, RT-PCR or NGS is selected from the group consisting of Taq- polymerase, Tog-polymerase and Tgo-polymerase. Most preferably, the polymerase used for PCR, RT-PCR or NGS is a Taq-polymerase.

[0082] "Polymerase chain reaction" (PCR), as used within the context of the present invention, is the technique in molecular biology, wherein a DNA polymerase is used to amplify a DNA fragment by enzymatic replication in vitro, which is well known to the person skilled in the art.

[0083] The present invention further relates to the use of a solution comprising a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group for removing SDS and / or LiDS from a biological sample. It is preferred for the use of the present invention that the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof. In one embodiment of the use of the present invention, the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-octanol. In one embodiment of the use of the present invention, the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol. In one embodiment of the use of the present invention, the C8-C12-alkane comprising at least one endterminal hydroxyl-group is 1-dodecanol.

[0084] In a further aspect, the present invention relates to a kit for removing SDS and / or LiDS from a biological sample, comprising(i) a C8-C12-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8- Ci2-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1- decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, and(ii) at least one component(s) selected from the group consisting of a lysis buffer, an agent for enzymatic digestion, preferably a protease, RNase or DNAse, a buffer, MgCI2solution and a size exclusion chromatography resin.

[0085] In one preferred embodiment of the kit of the present invention, the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-octanol. In one preferred embodiment of the kit of the present invention, the C8-C12-alkane comprising at least one end-terminal hydroxylgroup is 1-decanol. In one preferred embodiment of the kit of the present invention, the C8-C12- alkane comprising at least one end-terminal hydroxyl-group is 1-dodecanol.* * *

[0086] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0087] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time, etc.) that can be up to 5 %, up to 10 % of the given value. For example, if a formulation comprises about 5 mg / ml of a compound, this is understood to mean that a formulation can have between 4.5 and 5.5 mg / ml.

[0088] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent, the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0089] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0090] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term"comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[0091] When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0092] In each instance herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0093] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0094] The invention is further characterized by the following items:1. A method for isolating nucleic acid from a biological sample, comprising:(a) bringing the biological sample into contact with:• a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and• a C8-Ci2-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase,(b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about 90°C;(c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and(d) directly transfer of the aqueous phase onto a size exclusion chromatography resin.2. Method of item 1, wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, preferably wherein the C8- Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol.3. Method of item 1 or 2, wherein step (a) is conducted in absence of (a) chaotropic agent(s).4. Method of item 1 or 2, wherein the lysis buffer does not comprise (a) chaotropic agent(s).5. Method of any one of the preceding items, wherein the method is without a SDS and / or LiDS precipitation step.6. Method of any one of the preceding items, wherein the method is without the presence of monovalent ions of alkali metals and / or divalent ions of alkaline earth metals, preferably selected from the group consisting of Rb+, Cs+, Ca2+, Sr2+and Ba2+.7. Method of any one of the preceding items, wherein step (b) is carried out for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, preferably for at least 5 minutes.8. Method of any one of the preceding items, wherein the biological sample is a wax-embedded biological sample.9. Method of item 8, wherein the wax-embedded biological sample is a formalin-fixed paraffin- embedded (FFPE) biological sample.10. Method of item 9, wherein the FFPE biological sample is a FFPE feces sample, a FFPE cell culture sample, a FFPE blood sample, a FFPE urine sample, a FFPE tissue sample or a FFPE body fluid sample.11. Method of item 9 or 10, wherein bringing the biological sample into contact with the C8-C12- alkane comprising at least one end-terminal hydroxyl-group is after the paraffin of the FFPE biological sample is dissolved.12. Method of any one of the preceding items, wherein the method further comprises (an) enzymatic digestion step(s) at a temperature of at least 60°C, preferably comprising mixing, preferably for about 30 minutes.13. Method of any one of the preceding items, wherein at least one enzyme(s) is / are used for the enzymatic digestion step(s).14. Method of item 13, wherein the at least one enzyme(s) is / are lytic enzyme(s).15. Method of item 13 or 14, wherein the at least one enzyme(s) is / are selected from the group consisting of proteases, lipases, cellulases, hydrolases, chitinases, amylases and glucanases, preferably wherein hydrolases are not nucleases.16. Method of any one of items 13 to 15, wherein the at least one enzyme(s) comprise(s) at least one protease(s).17. Method of item 16, wherein the at least one protease(s) is / are selected from the group consisting of protease from Bacillus licheniformis, protease from Bacillus spec., protease from Staphylococcus aureus, protease from Bacillus amyloliquefaciens, protease from Coprinus spec, and protease from Aspergillus oryzae.18. Method of any one of the preceding items, wherein said size exclusion chromatography resin of step (d) is a size exclusion chromatography resin with exclusion limits in the range of 20 to 2000 bp of single and / or double stranded nucleotide strands.19. Method of any one of the preceding items, wherein said size exclusion chromatography resin is incorporated into a spin column.20. Method of any one of the preceding items, wherein the nucleic acids of the biological sample are ribonucleic acids (RNA) or desoxyribonucleic acids (DNA), preferably DNA.21. Method of any one of the preceding items, wherein step (a) is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes, preferably for at least 40 minutes.22. Method of any one of the preceding items, wherein SDS and / or LiDS is / are present in step (a) at a concentration in the range of about 10 mM to about 200 mM, preferably at a concentration in the range of about 20 mM to about 150 mM, more preferably at a concentration in the range of about 50 mM to about 100 mM.23. Method of any one of the preceding items, wherein the method further comprises (an) enzymatic digestion step(s) at a temperature of at least 60°C, preferably comprising mixing, preferably for about 30 minutes.24. Use of a solution comprising a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-C12- alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, for removing SDS and / or LiDS from a biological sample.25. A kit for removing SDS and / or LiDS from a biological sample, comprising(i) a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8- C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1- decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, and(ii) at least one component(s) selected from the group consisting of a lysis buffer, an agent for enzymatic digestion, preferably a protease, RNase or DNAse, a buffer, MgCI2solution and a size exclusion chromatography resin.EXAMPLES OF THE PRESENT INVENTION

[0095] MATERIALS AND METHODS

[0096] Preparation of FFPE samples

[0097] The fixation of tissue in FFPE is a widely used and valuable process to preserve tissues. However, no standard protocol exists despite its big influence on the quality and outcome for downstream applications. One reason is that different preparation methods might be needed and that several factors have influence on the tissue and preservation. The larger the biopsy, the more inhomogeneous the tissue preservation can be. The time-point when the biopsy is embedded in paraffin is also not standardized. A delay can lead to degradation of the tissue, proteins and nucleic acids among others. The longer a biopsy is preserved in formalin, the greater the crosslinking. To reverse this crosslinking, incubation at 90 °C is carried out. The sample is then incubated for 40 minutes. For FFPE tissue with high crosslinking, an extension of the incubation to 60 minutes can lead to a more efficient de-crosslinking resulting in higher yields. However, prolonging the incubation at 90 °C can cause higher fragmentation of the DNA.

[0098] DNA fragmentation

[0099] Formalin fixation and paraffin embedding crosslinks cell components, such as DNA, RNA, proteins and enzymes to stop cell metabolism. The longer the FFPE sample is stored, the higher thedegree of the resulting nucleic acid fragmentation. Furthermore, fragmentation happens at a higher rate at higher temperatures. Thus, on one hand, higher temperatures during the embedding process as well as during the de-crosslinking step increase the degree of fragmentation. On the other hand, these higher temperatures can increase the nucleic acid yield. For optimal DNA extraction, the time of the de-crosslinking incubation should take into consideration the sample age and length of formalin fixation, both of which also have an impact on fragmentation. For samples that have been stored several months or years or that have been fixed longer than needed for the used tissue size, extending the incubation at 90 °C from 40 minutes to 60 minutes can lead to a more efficient decrosslinking and results in higher yields.[001001 Excessive paraffin content

[0101] Large amounts of paraffin contained in a sample can make handling difficult and has a negative impact on yield and quality of DNA. For such samples, a reduction of the lysate volume and eluate volume is also to be expected. During sample preparation, as much surrounding paraffin as possible should be removed and only the "usable area" (see Figure 1) should be used for DNA extraction. This is especially recommended for DNA extraction of small FFPE tissue biopsies contained in a large piece of paraffin.

[0102] EXAMPLE 1:

[0103] Example 1 displays an exemplary way for carrying out the method of the present invention, however, is not intended to be limiting in any way. This method has been developed to extract / isolate for example total DNA from biological samples, but can also be performed for extraction / isolation of RNA. Further, said method is intended for any biological sample. It can also be used for a biological sample, which is a formalin-fixed paraffin-embedded (FFPE) tissue. Anyway, said method according to the present invention is not based on a silica-technology.1) Preparation before starting:• Vortex spin columns thoroughly to homogenize the matrix and remove air bubbles.• Then place them in a 2 mL reaction tube and let them stand to sediment the resin until step 6) "DNA purification".• Heat the thermomixer to 90 °C; if available, heat a second thermomixer to 60 °C.• Set microcentrifuge to 1,000 x g.• Prepare 1.5 mL microcentrifuge tubes for each sample.[0010412) FFPE tissue transfer, if biological sample is a FFPE biological sample:• When using FFPE blocks, cut sections using a microtome. Use of FFPE sections on slides is also possible. For both FFPE blocks and FFPE sections avoid transferring surrounding paraffin (see Figure 1).• For each sample, use 1 - 15 mg FFPE tissue or a minimum of 100 mm2and not more than 800 mm2of sections up to 10 pm thickness. Transfer FFPE tissue into a 1.5 mL microcentrifuge tube.• An excessive amount of surrounding paraffin can negatively influence the purification process resulting in reduced yield. Therefore, remove as much surrounding paraffin as possible. Genomic DNA yield is influenced by the FFPE tissue type and storage time. To ensure adequate yield from different FFPE tissues, adjust the amount of starting material accordingly.• Sections thicker than 10 pm can result in lower yields due to inefficient removal of paraffin.[0010513) FFPE de-crosslinking, if biological sample is a FFPE biological sample:• Add 110 pL lysis buffer comprising 70 mM SDS to each sample.• Incubate samples at 90 °C for 40 min in the thermal shaker with constant shaking at minimum 1,400 rpm (or maximum) for de-crosslinking. The degree of crosslinking in FFPE tissue depends on how long the fresh tissue was fixed in formaldehyde. For samples with a high degree of crosslinking, extending the incubation to 60 minutes at 90 °C can lead to a more efficient decrosslinking, resulting in higher yields. However, extending the incubation time can lead to higher fragmentation of DNA.• Remove tube from thermal shaker and let cool down to room temperature (around 5 minutes).[0010614) FFPE tissue lysis, detergent and paraffin removal:• Add 10 pL protease subtilisin directly into the sample.• Add 200 pL of 1-decanol solution to the reaction mix.• Incubate at 60 °C for 30 min in a thermal shaker with constant shaking at minimum 1,400 rpm.• Incubate at 90 °C for 5 min in a thermal shaker with constant shaking of 1,400 rpm (or maximum).• Proceed with step 5) during incubation.• Extending the incubation time at 90 °C to 10 minutes can improve results depending on the tissue and age of the sample. However, extending the incubation time can also lead to more fragmentation of DNA.[0010715) Spin column preparation:• Loosen the cap of the spin column % a turn and snap off the bottom.• Place the spin column in a 2 mL reaction tube.• Centrifuge 1 min at 1,000 x g, and discard flow-through.• Place the spin column in a fresh 1.5 mL reaction tube.[0010816) DNA purification:• After incubation, centrifuge the tubes from step 3 for 2 min at maximum speed (minimum 10,000 x g) to separate the aqueous (lower) and hydrophobic (upper) phases.

[0109] 0ptional for RNase digestion:• Transfer the lower aqueous phase (lysate) into a new 1.5 mL microcentrifuge tube.• Add 1 pL RNase into lysate and homogenize inverting several times or by vortexing the sample briefly.• Incubate 2 min at room temperature.

[0110] • Transfer 80 pL from the lower aqueous phase (lysate containing the DNA) onto the prepared spin column.• Close the cap of the spin column and loosen the cap again by % a turn.• Centrifuge the loaded column for 1 min at 1,000 x g.• Purified DNA is in the flow-through.The extracted DNA can be stored or used directly. DNA can be stored at 4 °C (short term) or at 20 °C (long term).The single-step purification of the method of the present invention is further depicted herein in Figure 2.[001111 EXAMPLE 2: Removal of SDS

[0112] This Example aimed at investigating the influence of alcohols and alkanes for their ability to remove SDS from a solution:• 110 pL of a reference buffer with a SDS concentration of 70 mM were placed in a reaction vessel.• Then 200 pL of 1-decanol, 1-octanol, 1-dodecanol and hexadecane were added. A batchwithout any other chemical was used as a reference.• In addition, 110 pL of a 140 mM SDS solution were placed in a reaction vessel and 200 pL of 1-decanol were added.• 3 replicates were made from each of these batches. • The reaction vessels were then incubated for 40 minutes at 1400 rpm and 90°C on the thermal shaker.• The reaction vessels were then centrifuged at 21000 x g for 2 minutes and a SDS test was carried out with the lower aqueous phase (after dilution).

[0113] The following results have been obtained by this Example:

[0114] Table 1: Absorbtion at 438 nm

[0115] Table 2: Like Table 1, but not diluted (absorbtion at 438 nm x dilution factor)

[0116] Table 3:

[0117] Table 4:

[0118] Table 5:

[0119] Table 6:

[0120] Those results showed that with the use of any of 1-decanol, 1-octanol or 1-dodecanol, the amount of SDS could be significantly reduced, at least more than 95% for 1-dodecanol and even at least 99% for both 1-decanol and 1-octanol. However, no reduction of the amount of SDS could be achieved by the use of hexadecane.

[0121] Those results have been obtained under consideration of the following standard curve as depicted in Figure 3. The respective values are given herein below in Table 7.

[0122] Table 7:rooi231 EXAMPLE 3:

[0124] A standard BE preparation for FFPE tissue was carried out as follows:• Weigh in FFPE tissue,• Pipette 110 pl lysis buffer into the reaction vessel and incubate for 40 minutes at 90°C in a head shaker at 1400 rpm,• Then allow to cool to room temperature,• Add 10 pl tissue protease and then add 200 pl 1-decanol,• Then incubate the mixture for 30 min at 60°C on the headshaker at 1400 rpm,• Then incubate directly for 5 minutes at 90°C,• After this step, the mixture is stirred for 2 minutes and centrifuged at max. speed in the tabletop centrifuge,• Then, the lower phase is removed and pipetted onto a previously conditioned S200 column (filling 630 pl),• Centrifugation for 1 min at 1000 x g.The SDS test of Example 3 was carried out to detect the reduction of SDS amount. The lysate of the respective sample was investigated. This was the lower phase after centrifugation at max. speed. The lysates were not purified via the column in Example 3.The following samples have been investigated and the results thereof are given in Figure 4 asdepicted herein.• Sample 1: An FFPE BE preparation without protease, with presence of 1-decanol and without S200 purification was carried out.• Sample 2: An FFPE BE preparation with presence of 1-decanol and without S200 purification was carried out.• Sample 3: An FFPE BE preparation with presence of 1-decanol and without S200 purification was carried out.• Sample 4: Like sample 2 and 3 with presence of 1-decanol, only at the end 10 minutes at 90°C, instead of 5 minutes.• Sample 5: Like sample 2 and 3 with presence of 1-decanol, only at the end 15 minutes at 90°C, instead of 5 minutes.• Sample 6: An FFPE BE preparation with presence of 1-decanol was carried out without any heating-step, but at RT, and without S200 purification. It was shaken at 1400 rpm for the same amount of time as for a normal preparation.• Sample 7: Pure tissue lysis buffer was tested (70 mM SDS).• Sample 8: A BE preparation was carried out without tissue and without protease. Only hexadecane and tissue lysis buffer were combined as described above.• Sample 9: 110 pL TE was mixed with 10 pL tissue protease and the SDS test was carried out directly after mixing to show that the reagent did not react with proteins and therefore resulted in a color change.• Sample 10: Incubate tissue lysis buffer with 200 pL 1-decanol for 5 minutes at 90°C on the head-shaker at 1400 rpm, without S200 purification.• Sample 11: Incubate tissue lysis buffer with 200 pL 1-decanol for 10 minutes at 90°C on the head-shaker at 1400 rpm, without S200 purification.• Sample 12: Incubate tissue lysis buffer with 200 pL 1-decanol for 15 minutes at 90°C on the head-shaker at 1400 rpm, without S200 purification.• Sample 13: TE directly applied to the SDS test as a second blank in addition to water.

[0125] Those results have been obtained under consideration of the following standard curve as depicted in Figure 5. The respective values are given herein below in Table 8.

[0126] Table 8:

[0127] The results shown in Fig. 4 lead to the following conclusions:

[0128] The concentrations determined for samples 1, 2 and 3 showed that the introduction of tissue positively influences the effect of SDS depletion. Samples 4 and 5 showed that extending the 90°C- step at the end of the preparation also increases SDS depletion. Sample 6 showed that SDS depletion without temperature steps does not work with 1-decanol. As a reference, sample 7 is used, where the SDS concentration of the untreated TLB was measured. Since the measured absorbances at 438 nm were clearly outside the calibration curve, the real concentration was significantly higher than given here. This was 70 mM. More measurement data can be seen in example 2. Thus, it can be deduced that no SDS depletion took place in samples 6 and 8. With sample 8, it can be seen that with hexadecane as liquid organic phase, despite the same sample treatment as samples 1-5, no SDS depletion took place. To test whether the protease has an influence on the measurement method, these were measured together with a TE buffer in sample 9. Both were mixed together in the same ratio as in a BE preparation. The measurement of sample 9 showed that the protease has no influence on the measurement method. Samples 10, 11 and 12 showed that the 90°C tempering in combination with the shaking on the headshaker provide for SDS depletion. It is also shown that prolonging the incubation at 90°C results in increased SDS depletion. Sample 13 showed that, similar to sample 9, the TE buffer has no influence on the measurement of SDS concentration.

Claims

CLAIMS1. A method for isolating nucleic acid from a biological sample, comprising:(a) bringing the biological sample into contact with:• a lysis buffer comprising SDS and / or LiDS to form an aqueous phase, and• a C8-C12-alkane comprising one end-terminal hydroxyl-group to form a liquid organic phase,(b) mixing the two phases of step (a) at a temperature in the range between about 80°C and about90°C;(c) allow the mixture of step (b) to form a multiple phase mixture comprising at least the two phases of step (a); and(d) directly transfer of the aqueous phase onto a size exclusion chromatography resin.

2. Method of claim 1, wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol or 1-dodecanol, preferably wherein the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol.

3. Method of claim 1 or 2, wherein step (a) is conducted in absence of (a) chaotropic agent(s).

4. Method of any one of the preceding claims, wherein the method is without a SDS and / or LiDS precipitation step.

5. Method of any one of the preceding claims, wherein the method is without the presence of monovalent ions of alkali metals and / or divalent ions of alkaline earth metals, preferably selected from the group consisting of Rb+, Cs+, Ca2+, Sr2+and Ba2+.

6. Method of any one of the preceding claims, wherein step (b) is carried out for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, preferably for at least 5 minutes.

7. Method of any one of the preceding claims, wherein the biological sample is a wax-embedded biological sample.

8. Method of claim 7, wherein the wax-embedded biological sample is a formalin-fixed paraffin- embedded (FFPE) biological sample,preferably wherein the FFPE biological sample is a FFPE feces sample, a FFPE cell culture sample, a FFPE blood sample, a FFPE urine sample, a FFPE tissue sample or a FFPE body fluid sample.

9. Method of claim 8, wherein bringing the biological sample into contact with the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is after the paraffin of the FFPE biological sample is dissolved.

10. Method of any one of the preceding claims, wherein the method further comprises (an) enzymatic digestion step(s) at a temperature of at least 60°C, preferably comprising mixing, preferably for about 30 minutes.

11. Method of any one of the preceding claims, wherein at least one enzyme(s) is / are used for the enzymatic digestion, preferably wherein the at least one enzyme(s) is / are selected from the group consisting of proteases, lipases, cellulases, hydrolases, chitinases, amylases and glucanases.

12. Method of claim 11, wherein the at least one enzyme(s) comprise(s) at least one protease(s), preferably wherein the at least one protease(s) is / are selected from the group consisting of protease from Bacillus licheniformis, protease from Bacillus spec., protease from Staphylococcus aureus, protease from Bacillus amyloliquefaciens, protease from Coprinus spec, and protease from Aspergillus oryzae.

13. Method of any one of the preceding claims, wherein said size exclusion chromatography resin of step (d) is a size exclusion chromatography resin with exclusion limits in the range of 20 to 2000 bp of single and / or double stranded nucleotide strands, preferably wherein said size exclusion chromatography resin is incorporated into a spin column.

14. Use of a solution comprising a C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8-C12-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-Ci2- alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, for removing SDS and / or LiDS from a biological sample.

15. A kit for removing SDS and / or LiDS from a biological sample, comprising(i) a C8-C12-alkane comprising at least one end-terminal hydroxyl-group, preferably wherein the C8- Ci2-alkane comprising at least one end-terminal hydroxyl-group is selected from 1-octanol, 1-decanol, 1-dodecanol or mixtures thereof, more preferably wherein the C8-Ci2-alkane comprising at least one end-terminal hydroxyl-group is 1-decanol, and(ii) at least one component(s) selected from the group consisting of a lysis buffer, an agent for enzymatic digestion, preferably a protease, RNase or DNAse, a buffer, MgCI2solution and a size exclusion chromatography resin.

Images