Methods for isolating and / or purifying nucleic acids

Abstract

The present disclosure refers to methods for isolating and / or purifying nucleic acids from a sample, kits for carrying out the methods for isolating and / or purifying nucleic acids, and formulations for use in the methods for isolating and / or purifying nucleic acids.

Description

[Technical Field]

[0001] The present invention relates to methods for isolating and / or purifying nucleic acids from a sample, kits for carrying out said methods for isolating and / or purifying nucleic acids, and formulations for use in said methods for isolating and / or purifying nucleic acids. [Background technology]

[0002] Analysis of nucleic acids, such as ribonucleic acid (RNA) and desoxyribonucleic acid (DNA), is of great importance in biological, agricultural, biotechnology, medical, and pharmaceutical research and diagnostics. For example, examination of cellular nucleic acids allows the determination of a cell's genetic origin and functional activity. In addition, genetic markers for the detection and / or prediction of disease or phenotypic traits can also be identified. Furthermore, analysis of RNA and DNA is also useful for identifying pathogenic bacteria, fungi, and viruses. Important, non-limiting examples are the nucleic acids of pathogens, such as viral nucleic acids. Thus, extracellular nucleic acids are particularly useful in non-invasive diagnosis and prognosis (prognosis) and can be used as diagnostic markers in many fields of application, such as non-invasive prenatal genetic testing, oncology, transplantation medicine, or many other diseases, and are therefore suitable for diagnosis.

[0003] However, many of these methods for analyzing nucleic acids require nucleic acids as a substrate that has been essentially isolated and purified from any other cellular components and further contaminants introduced into the sample during previous steps, e.g., the lysis step. Therefore, high-throughput methods for nucleic acid isolation and / or purification are desirable, allowing for fast and reliable nucleic acid isolation and / or purification, preferably in an environmentally friendly manner.

[0004] A variety of methods are known for isolating nucleic acids, such as DNA and RNA, from aqueous samples containing the nucleic acids, for example biological samples dissolved in an aqueous lysis buffer.

[0005] In the areas of molecular diagnostics and modern genetic breeding efforts, the steadily increasing number of samples is creating bottlenecks in workflows because either the assays for nucleic acid analysis or the isolation of nucleic acids to achieve sufficient purity for analysis are too slow or too costly.

[0006] Typically, nucleic acid extraction ("crude lysis"), such as HotShot or QuickExtract® (LGC Biosearch Technologies Ltd.), is used, which allows for the rapid release of nucleic acids from sample materials and their direct use in subsequent analysis. The drawback of this approach is that assay inhibitors are not removed but are diluted, reducing the amount of nucleic acid available for assay (reducing concentration). Furthermore, nucleic acids are often degraded due to harsh processing conditions. Additionally, different methods are required for different sample types, which cannot be streamlined, and therefore a lot of effort is required to find suitable dilutions and conditions for each sample type. Additionally, the error rate in assays is very high. With the advent of more expensive downstream assays, such as next-generation sequencing, crude methods are often no longer acceptable.

[0007] Proper purification of nucleic acids is often slow because the reagents used to dissolve the sample and bind the nucleic acids to the solid support interfere with downstream assays, necessitating the need to wash away and remove the reagents prior to downstream analysis. Typically, nucleic acid purification using solid supports is completed in the following order: dissolution, binding, two to three washing steps, drying, and elution. This often results in kits that are expensive because they use a lot of plastic during processing, generate a lot of environmentally hazardous waste, and require a large amount of chemicals for purification. Additionally, each step increases the time from sample to result.

[0008] Several approaches have been attempted to overcome this problem, for example, US 2001 / 0018513 A1 relates to the use of a solid support comprising a plurality of positively ionizable groups immobilized on the solid phase, where nucleic acids are bound at a pH where the ionizable groups are positively charged, the nucleic acids are washed with an aqueous buffer below pH 7, and the nucleic acids are released at pH values ​​where the ionizable groups are less likely to be negatively, neutrally, or positively charged.

[0009] US 2013 / 0158247A1 discloses the use of positively charged binding ligands containing at least one basic moiety and / or quaternary ammonium group, which bind negatively charged nucleic acids at a first pH before or during contact with a solid support bearing an acidic moiety, e.g., a carboxy, sulfonic acid, phosphinic acid, or acidic hydroxyl group, followed by washing with an aqueous, low-salt buffer and releasing the nucleic acid using an elution buffer at a second pH lower than the first pH and / or a pH greater than 7 if the ligand contains a quaternary ammonium group.

[0010] China Patent No. 113584019 A discloses a reagent and kit for rapid extraction of nucleic acids from FFPE samples, as well as uses of the reagent and kit. The binding buffer contains 1-5 M guanidine, Tween 20 (1-2% v / v), polyethylene glycol octylphenyl ether (Triton X-100, 1-5% (v / v)), EDTA (10-40 mM), and Tris-HCl (20-60 mM) at a pH between 8.0 and 9.5, creating a binding mixture to which nucleic acids are absorbed (adsorbed). Reagents for rapid extraction of nucleic acids from FFPE samples also include magnetic beads. However, this method has the disadvantages of requiring a very specific principle and washing with an organic solvent, as the method operates at a basic pH and requires a guanidine salt to adsorb nucleic acids onto a solid support.

[0011] German Patent Application No. DE 10 2007 009 347 A1 discloses a method using adsorption of nucleic acids to polar, weakly acidic surfaces using alcohols, chaotropic salts, and ionic detergents, or mixtures thereof, followed by washing of the polar, bound nucleic acids with an aqueous buffer containing a mediator with primary, secondary, or tertiary ammonium groups. The positively charged mediator indirectly tethers the negatively charged nucleic acids to the solid support and releases the nucleic acids under pH conditions above pH 8. The drawback here is that the binding material requires washing steps before the mediator can be applied, and each washing step is time-consuming (5-10 minutes per washing step). Summary of the Invention [Problem to be solved by the invention]

[0012] A drawback of the methods described in the prior art is that, due to the nature of the binding, they are susceptible to interfering substances, such as anionic or cationic surfactants used for lysis, such as SDS (sodium dodecyl sulfate) or CTAB (cetyltrimethylammonium bromide), or guanidine salts used in the binding step or to keep proteins in solution. These often need to be precipitated or washed out of the sample before purification, which means additional time, effort, and expense. Furthermore, the methods described in the prior art still require multiple washing steps, especially when attempting to isolate nucleic acids from sample materials containing many secondary metabolites and other potential PCR inhibitors, such as plant sample materials, because the inhibitors are also bound to the solid support.

[0013] Therefore, there remains a need for a very fast and environmentally friendly DNA isolation method that results in high quality DNA.

[0014] It is an object of the present invention to provide a method for isolating and / or purifying nucleic acids from a nucleic acid-containing biological sample, which method avoids at least one of the drawbacks of the prior art discussed above. In certain embodiments, it is an object of the present invention to provide a simple and rapid method for isolating and / or purifying nucleic acids, which is suitable for automation and which can process a large number of samples. [Means for solving the problem]

[0015] The invention is defined by the following claims.

[0016] The present invention relates to a novel technology that uses a specific binding solution for the isolation and / or purification of nucleic acids. Therefore, the method according to the present invention is not based on known mechanisms for binding nucleic acids to solid phases, which typically use a combination of alcohols and divalent salts to bind DNA to solid phases or to bind negatively charged nucleic acids to positively charged surfaces (charge switch). Similarly, the method according to the present invention does not require chaotropic ("boom" chemistry) or kosmotropic salts to promote binding of nucleic acids to the silica surface of the solid phase. The present invention is particularly based on the surprising discovery that the binding solution used in the method according to the present invention, which is based on a combination of a nonionic detergent and, optionally but preferably, a protic alcohol on the solid phase, enables fast and environmentally friendly purification of nucleic acids without compromising the quality or yield of the nucleic acids. The method according to the present invention allows for the isolation and / or purification of nucleic acids independently of the detergent used for lysis. Therefore, the method according to the present invention is adaptable to almost any sample type, offering high flexibility.

[0017] According to a first aspect, the present invention provides a method for producing a cellular membrane comprising: a) providing a sample containing nucleic acids and binding the nucleic acids to a solid phase containing carboxyl and / or hydroxyl groups by means of a binding solution, The binding solution is i) at least one non-ionic surfactant in a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1~4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the binding mixture, comprising the binding solution and the sample containing nucleic acid, has a pH between 3 and 6 that allows the nucleic acid to bind to the solid phase; b) separating the solid phase with bound nucleic acids from the binding mixture; c) optionally washing the nucleic acid; and d) Eluting the nucleic acid from the solid phase The present invention provides a method for isolating and / or purifying nucleic acids from a sample, comprising:

[0018] According to a second aspect, a) lysis buffer, and b) a binding solution, i) at least one non-ionic surfactant at a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1~4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the binding buffer has a pH selected from 6 or less, 5.5 or less, 4 or less, and 3 or more. binding solution; c) a solid phase containing carboxyl and / or hydroxyl groups capable of selectively binding nucleic acids in the presence of said ligand compound; and d) optionally one or more cleaning solutions, and e) one or more elution solutions There is provided a kit for carrying out the method according to the first aspect, comprising:

[0019] According to one embodiment, binding solution b) is chaotropic salt-free and divalent cation-free. Thus, binding solution b) of the kit according to the invention does not use any chaotropic salts or divalent cations.

[0020] Further, other aspects of the present disclosure include: i) at least one non-ionic surfactant in concentrations of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v); ii) ethanol or isopropanol at a concentration of 50% to 75%; and iii) a ligand compound comprising at least one protonatable group, the ligand being selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or a corresponding salt thereof; and iv) Acidifying and / or buffering compounds to adjust the pH value of the formulation to between 3 and 6

[0023] In another aspect, the present invention relates to a formulation for use in a method according to the first aspect, comprising:

[0021] According to one embodiment, the formulation according to the invention is chaotropic salt-free and divalent cation-free.

[0022] Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the present application, are given by way of illustration only. Various changes and modifications within the scope of the disclosed invention will become readily apparent to those skilled in the art from the following teachings. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention This disclosure may embody various different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided as examples so that this disclosure will be thorough and complete, and will fully convey the aspects and features of the present disclosure to those skilled in the art.

[0024] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Furthermore, the use of "may," when describing embodiments of the present disclosure, refers to "one or more embodiments of the present disclosure." In the following description of embodiments of the present disclosure, singular terms may include plural forms unless the context clearly dictates otherwise.

[0025] As used herein, the terms "substantially," "about," and similar terms are used as terms of approximation rather than degree, and are intended to take into account inherent variations in measurements or calculations that would be recognized by one of ordinary skill in the art. Furthermore, when the term "substantially" is used in conjunction with a characteristic that can be expressed using a numerical value, the term "substantially" indicates a range of + / - 5% of the value centered on the value.

[0026] It may be further understood that the terms "include," "comprise," "including," or "comprising" specify properties, ranges, fixed values, steps, processes, elements, components, and combinations thereof, but do not exclude other properties, ranges, fixed values, steps, processes, elements, components, and combinations thereof.

[0027] The term "solution," particularly as used herein, refers to a liquid composition, preferably an aqueous composition. The solution may be a homogeneous mixture of only one phase, but it is also within the scope of the present invention for the solution to contain solid additives, such as precipitates, stabilizers, etc., especially those contained in chemicals.

[0028] The term "binding mixture," as used herein, refers to a composition comprising a nucleic acid-containing sample and a binding solution, which is prepared in a binding step to allow the nucleic acids contained in the sample to bind to a solid phase. The binding mixture thus provides suitable conditions for binding the nucleic acids to the solid phase.

[0029] basic concept The present invention provides the following: a) providing a sample containing nucleic acids and binding the nucleic acids to a solid phase containing carboxyl and / or hydroxyl groups by means of an aqueous binding solution, The binding solution is i) at least one non-ionic surfactant in a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1~4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the binding mixture, comprising the binding solution and the sample containing nucleic acid, has a pH between 3 and 6 that allows the nucleic acid to bind to the solid phase; b) separating the solid phase with bound nucleic acids from the binding mixture; c) optionally washing the nucleic acid; and d) Eluting the nucleic acid from the solid phase The present invention provides a method for isolating and / or purifying nucleic acids from a sample, comprising:

[0030] The method according to the invention represents a great advantage in that it is independent of the components used in the lysis buffer system and is therefore not negatively affected by anionic or cationic detergents, by chaotropic salts, or by high molar concentrations of kosmotropic salts. The method according to the invention is therefore adaptable to almost any type of lysis sample and therefore offers a high degree of flexibility.

[0031] The isolation and / or purification methods of the present invention have significant advantages over methods used in the prior art in that they are rapid, allow simple processing of large numbers of samples, require minimal handling steps, require no wash buffers other than water, which is readily available in any laboratory, provide high recovery and purity of extracellular nucleic acids, and are automatable. Thus, the methods of the present invention can provide customers with 3-8 times faster throughput without the need for equipment modifications and without sacrificing nucleic acid quality or yield. Additionally, the methods reduce the cost of consumables, reduce waste volume and toxic waste, and reduce transportation costs and storage requirements.

[0032] The nucleic acid(s) that can be isolated and / or purified using the methods of the present invention include DNA and RNA, particularly genomic DNA (gDNA), plasmid DNA, PCR fragments, cDNA, rRNA, mRNA, miRNA, cfDNA, siRNA, and oligonucleotides, from microorganisms (including viruses, bacteria, and fungi), humans, animals, or plants, as well as modified nucleic acids, such as so-called peptide nucleic acids or locked nucleic acids (PNA or LNA, respectively). In addition, hybrids formed from DNA and RNA can also be purified, but are not limited to these.

[0033] The sample to be processed by the method of the present invention may be any sample containing nucleic acids, and is preferably a biological sample, either in its natural state or in a processed form. Preferably, the sample may include body fluids, such as blood, serum, sputum, feces, plasma, semen, cerebrospinal fluid, saliva, human, animal, or plant tissues and tissue cultures, microorganisms, human, animal, or plant cells and cell cultures, and human or animal organs or parts thereof, such as liver, kidney, or lung. Additionally, the sample may be a fluid sample, such as, but not limited to, waste, drinking water, juice, or food. Additionally, the sample may be a processed biological sample, such as a human, animal, or plant cell lysate, a microbial lysate (bacterial lysate), paraffin-embedded tissue, an aqueous or buffered sample solution, or a gel.

[0034] If the nucleic acid to be isolated is present in cellular material, it may be preferable to first disrupt the cellular material by any method known in the art for releasing nucleic acid from cells, such as mechanical, chemical, or enzymatic lysis, before further processing the nucleic acid according to the method of the present invention. Preferably, the cellular material is lysed to release the nucleic acid, and a sample containing nucleic acid (also referred to herein as a "lysate") can be formed using known lysis buffers and methods, such as contacting with ionic and non-ionic detergents, hypotonic solutions of salts, proteases, chaotropic agents, or solvents, crushing the tissue, or adding other lytic enzymes. The lysis solution is preferably a lysis buffer and a lysis solution of the organic material as the sample.

[0035] In one embodiment, the sample containing nucleic acids comprises a lysis buffer.

[0036] Examples of lysis buffers are low-salt, anionic detergent-based buffers, such as sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, or lauryldodecyl sulfate, ethylenediaminetetraacetic acid (EDTA), or high-salt, cationic detergent-based buffers, such as cetyltrimethylammonium bromide (CTAB), which produce a solution containing nucleic acids (lysis solution).

[0037] In the binding step a) of the method, the nucleic acid-containing sample, preferably contained in a lysis buffer, is contacted with a solid phase and components of a binding mixture having a pH value between 3 and 6, preferably between 4 and 5, to adsorb the nucleic acid to the solid phase. The binding solution comprises: i) at least one non-ionic surfactant, preferably an alcohol ethoxylate or polysorbate, at a concentration of at least 5% (w / v), optionally iii) a ligand compound containing at least one protonatable group, and ii) optionally, but preferably, a linear or branched C 1~5 It comprises an aqueous solution of alcohol or acetone, preferably ethanol or isopropanol. Preferably, the binding solution comprises iv) an acidifying compound and / or a buffer compound to adjust the pH value to between 3 and 6, preferably between 4 and 5, more preferably between 4 and 4.5.

[0038] For example, the lysis solution can be added to the binding solution, or the binding solution can be added to the lysis solution, although it is preferred to add the lysis solution to the binding solution. It may also be possible to lyse the sample directly with a binding solution containing a lysis buffer.

[0039] The binding solution may contain a solid phase, or the solid phase may be added in a separate step. Optionally, one or more components i), iii), and iv) of the binding solution may already be present in the lysis solution. The amounts of components i), ii), and iii) provided below refer to the binding solution containing components i), ii), and iii). The amounts of components i), ii), and iii) that refer to the final binding mixture containing lysis solution and binding solution are provided separately.

[0040] An important feature of the present invention is the incorporation of i) at least one non-ionic surfactant in the binding solution.

[0041] The term "detergent" refers to an amphiphilic organic compound composed of a hydrophobic, nonpolar hydrocarbon portion (tail) and a hydrophilic, polar head group. The ionic character of the polar head group forms the basis for the broad classification of surfactants; surfactants can be ionic (charged either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups, but a net charge of zero).

[0042] In a preferred embodiment, the surfactant comprises i) at least one non-ionic surfactant or a combination of two or more surfactants of non-ionic nature.

[0043] Preferably, the non-ionic surfactant is a polysorbate.

[0044] "Polysorbate," sometimes referred to as "Tween®," refers to a class of amphiphilic, nonionic surfactants derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Polysorbates are commonly produced by ethoxylating sorbitan and then esterifying it with fatty acids. The ethoxylation process distributes a certain number of repeating polyethylene glycol units across the four different attachment sites (hydroxyl groups) of the sorbitan molecule, while leaving a sorbitan core. Thus, the term "polysorbate" refers, inter alia, to a mixture composed of chemical species that vary according to a specific distribution of polyethylene glycol units across the sorbitan's hydroxyl groups.

[0045] Polysorbates are generally named according to the following nomenclature: The first digit essentially indicates the esterified fatty acid (2 = lauric acid, 4 = palmitic acid, 6 = stearic acid, 8 = oleic acid, 12 = isostearic acid). The second digit indicates the type of esterification (0 = monoester containing 20 polyoxyethylene units, 1 = monoester containing 4 or 5 polyoxyethylene units, 5 = triester containing 20 polyoxyethylene units). The term "PS20" or "polysorbate 20" refers to polyoxyethylene (20) sorbitan monolaurate. "PS80" or "polysorbate 80" refers to polyoxyethylene (20) sorbitan monooleate.

[0046] Thus, in the binding mixture used in the method according to the invention, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, and mixtures thereof. In a preferred embodiment, the polysorbate is selected from the group consisting of polysorbate 20, polysorbate 60, polysorbate 80, and mixtures thereof. More preferably, the polysorbate is polysorbate 20 (Tween® 20).

[0047] Non-limiting examples of additional nonionic surfactants useful in the bonding mixtures provided herein include polyethylene glycol tert-octylphenyl ether (commercially available from Sigma-Aldrich as Triton® X-100), copolymers of ethylene oxide and propylene oxide, such as Tergitol® (e.g., polyethylene glycol trimethylnonyl ether (commercially available from Sigma-Aldrich as Tergitol® TMN-6)), alcohol ethoxylates (e.g., 2-ethylhexanol EO-PO (Registration Model No. 64366-70-7; Ecosurf® series, e.g., Ecosurf® EH9 (available from Dow Chemicals), or Tergitol® 15-S-7, Tergitol® 15-S-9 (available from Sigma-Aldrich)), alkoxylates or ethoxylates of fatty alcohols, such as secondary alcohol alkoxylates (e.g., Ecosurf® LF-45 (available from Dow Chemicals)), and the like. and (ii) a mixture of two or more of the foregoing, preferably an alcohol ethoxylate, such as Ecosurf® EH9, Tergitol® 15-S-7, or Tergitol® 15-S-9.

[0048] In a preferred embodiment, the non-ionic surfactant is selected from the group of polysorbates, such as polysorbate 20, and alcohol ethoxylates, such as Ecosurf® EH-9, Ecosurf® EH-6, Ecosurf® EH-9, Ecosurf® SA-7, Ecosurf® SA-9, Ecosurf® 15-S-9, Tergitol® 15-S-7, Tergitol® 15-S-9, preferably Ecosurf® EH-9 or Tergitol® 15-S-7, more preferably Tergitol® 15-S-7.

[0049] The amount of non-ionic surfactant in the binding solution used in the method according to the present invention can vary. For example, the binding mixture can contain a non-ionic surfactant at a concentration selected from 5% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v). Particularly preferred is a concentration selected from 20% (w / v) to 30% (w / v).

[0050] In one embodiment, the amount of non-ionic surfactant in the binding solution is greater than 10% (w / v), more preferably between 15% and 25% (w / v).

[0051] The final concentration of the nonionic surfactant in the resulting binding mixture is 2.5% (w / v) to 40%, 5% (w / v) to 30% (w / v), 10% (w / v) to 25% (w / v), and 12.5% ​​to 15%. Particularly preferred is a final concentration selected from 10% (w / v) to 15% (w / v). The presence of a nonionic surfactant at a pH value between 3 and 6, preferably between 4 and 5.5, introduces cleaning properties and helps drive the nucleic acid to the solid phase. Subsequently, the bound nucleic acid may be optionally washed with a short water wash (pH less than 7) and then eluted from the solid phase using a basic buffer solution at pH 7.5 to 13.

[0052] The binding solution according to the present invention further comprises ii) an optional aqueous solution comprising a linear or branched C1-C4 alcohol, such as methanol, ethanol, n-propanol, isopropanol, or acetone, preferably ethanol or isopropanol, more preferably isopropanol, in a concentration of 0-90% (v / v). According to one embodiment, the binding solution according to the invention further comprises ii) methanol, ethanol, n-propanol, isopropanol or acetone, preferably ethanol or isopropanol, in a concentration selected from 15% to 90%, preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% (v / v), more preferably 40%, 45%, 50%, 55%, 60%, 65%, 70% (v / v), most preferably 50%, 55% and 60% (v / v).

[0053] Preferably, the binding solution according to the invention comprises isopropanol or ethanol, preferably isopropanol, in a concentration of 50% to 75%, preferably 55% to 70%, more preferably 60% to 65% (v / v).

[0054] The final concentration of linear or branched C1-C4 alcohol or acetone in the conjugation mixture is preferably between 5% and 70% (v / v), preferably between 10% and 35% (v / v), more preferably between 20% and 30% (v / v). According to a preferred embodiment, the final concentration is selected from 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% (v / v), more preferably 10%, 15%, 20%, 25%, 30%, 35%, 40% (v / v), and most preferably 20%, 25% and 30% (v / v).

[0055] According to one embodiment, the binding solution according to the present invention comprises i) at least one non-ionic surfactant at a concentration selected from 5% (w / v) to 70% (w / v), 8% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v), and ii) ethanol or isopropanol. Preferably, the non-ionic surfactant is polysorbate 20 or an alcohol ethoxylate, such as Ecosurf® EH-9, Ecosurf® EH-6, Ecosurf® EH-9, Ecosurf® SA-7, Ecosurf® SA-9, Ecosurf® 15-S-9, Tergitol® 15-S-7, Tergitol® 15-S-9, preferably Ecosurf® EH-9 or Tergitol® 15-S-7, more preferably Tergitol® 15-S-7.

[0056] According to a preferred embodiment, the binding solution according to the present invention comprises i) polysorbate 20 at a concentration selected from 10% (w / v) to 30% (w / v), and ii) ethanol or isopropanol at a concentration selected from 50%, 55%, and 60% (v / v).

[0057] We found that the use of protic alcohol or acetone in the binding solution allowed for the addition of 5–40% (w / v) nonionic surfactants without excessive viscosity buildup, which could affect pipetting, thus facilitating automation. Furthermore, the addition of alcohol is required for lysis solutions containing cationic surfactants, such as CTAB, a surfactant commonly used in plant DNA purification, to retain DNA on the beads. The presence of a nonionic surfactant not only promotes binding of nucleic acids to the solid support, but also introduces detergent properties to the binding mixture, allowing a single, short water wash step to adequately remove impurities.

[0058] This embodiment has significant advantages: in some downstream assays, it is possible to skip the optional washing step, remove the buffer used in the binding reaction, and directly elute the nucleic acids from the solid support. Surprisingly, the presence of a non-ionic detergent is important not only for binding the nucleic acids to the solid support in the presence of high molar salt and / or cationic detergent, but also for keeping the nucleic acids on the beads during the optional washing step with water.

[0059] The binding solution used in the method according to the present invention optionally contains iii) a ligand compound containing at least one protonatable group. The protonatable group is preferably a chemical group that is neutral or uncharged at high pH values ​​and protonates at low pH values, thereby having a positive charge. In particular, the protonatable group is positively charged at the pH at which the binding of nucleic acid to the particle occurs. Preferably, the ligand compound may contain one or more protonatable groups per molecule, preferably two or more, and most preferably three or more.

[0060] Examples of suitable protonatable groups include, in particular, amino groups, such as primary, secondary, and tertiary amino groups, as well as cyclic amines, aromatic amines, and heterocyclic amines. The amino group preferably has alkyl, alkenyl, alkynyl, and / or aromatic substituents (including cyclic substituents and substituents that, together with the nitrogen atom, form a heterocyclic or heteroaromatic ring). The substituents preferably contain 1 to 20 carbon atoms, more preferably 1 to 12, 1 to 8, or 1 to 6 carbon atoms. The substituents may be linear or branched and may contain heteroatoms, such as oxygen, nitrogen, sulfur, silicon, and halogen (e.g., fluorine, chlorine, bromine) atoms. Preferably, the substituents contain no more than 6, preferably no more than 5 or no more than 4 heteroatoms.

[0061] According to one embodiment, the ligand compound comprising at least one protonatable group is a primary, secondary, tertiary, or quaternary monoamine or polyamine, or an imine having at least one, preferably three or more, amino groups. Preferably, the ligand compound may comprise at least two primary amines, or at least one secondary or tertiary amine. Examples of suitable ligand compounds are ethylenediamine, diethylenetriamine, triethylenetetramine (TETA), tetraethylenepentamine, pentaethylenehexamine, trimethylamine (TMA), triethylamine (TEA), triethylaminoethyl (TEAE), diethanolamine, diethylamine, diisopropylamine, linear or branched polyethyleneimine (PEI), carboxylated or hydroxyalkylated polyethyleneimine, jeffamine, spermine, spermidine, poly(amidoamine) (PAMAM) dendrimers, polyallylamine, polyvinylamine, N-morpholinoethyl, polylysine, polyarginine, protamine, or tetraaza-cycloalkanes, and their corresponding salts.

[0062] According to a preferred embodiment, iii) the ligand compound comprising at least one protonatable group may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, pentaethylenehexamine, triethylamine, or linear or branched polyethyleneimine (PEI), carboxylated or hydroxyalkylated polyethyleneimine, and their corresponding salts. More preferably, the ligand compound may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or their corresponding salts. The use of tetraethylenepentamine or its corresponding salt is particularly preferred.

[0063] Diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine is used at a concentration within the range of 0.01 to 100 mM, preferably within the range of 0.1 to 60 mM, more preferably within the range of 0.2 to 50 mM, and even more preferably within the range of 1 to 40 mM.

[0064] According to one embodiment, the binding solution according to the present invention comprises: i) at least one non-ionic surfactant at a concentration selected from 5% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v); ii) ethanol or isopropanol; and iii) a ligand compound comprising at least one protonatable group, which may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine. Preferably, the concentration of diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine is in the range of 0.01 to 100 mM, preferably in the range of 0.1 to 60 mM, more preferably in the range of 0.2 to 50 mM, and even more preferably in the range of 1 to 40 mM.

[0065] According to one embodiment, the binding solution according to the invention comprises i) one non-ionic surfactant at a concentration of 20% (w / v) or 30% (w / v), and ii) isopropanol or ethanol at a concentration selected from 50%, 55%, and 60% (v / v), and iii) a ligand compound comprising at least one protonatable group at a concentration of 0.1 to 40 mM, which ligand compound may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or their corresponding salts, wherein i) the non-ionic surfactant is polysorbate 20 or alcohol ethoxylate. Preferably, the nonionic surfactant is an alcohol ethoxylate, such as Ecosurf® EH-9, Ecosurf® EH-6, Ecosurf® EH-9, Ecosurf® SA-7, Ecosurf® SA-9, Ecosurf® 15-S-9, Tergitol® 15-S-7, Tergitol® 15-S-9, and more preferably, the nonionic surfactant is an alcohol ethoxylate, such as Ecosurf® EH9 or Tergitol® 15-S-7.

[0066] According to a preferred embodiment, the binding solution according to the present invention comprises i) polysorbate 20 at a concentration of 20% (w / v) or 30% (w / v), ii) isopropanol or ethanol at a concentration selected from 50%, 55%, and 60% (v / v), and iii) a ligand compound containing at least one protonatable group, which may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or a corresponding salt thereof. Preferably, the concentration of diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine is in the range of 0.01 to 100 mM, preferably in the range of 0.1 to 60 mM, more preferably in the range of 0.2 to 50 mM, and even more preferably in the range of 1 to 40 mM.

[0067] A solid phase that binds nucleic acids is used in the methods according to the invention. In one embodiment of the methods according to the invention, the binding mixture and the solid phase containing carboxyl and / or hydroxyl groups are mixed, preferably for at least 30 seconds, more preferably for 2 minutes, before separating the solid phase from the binding mixture.

[0068] Examples of solid phase materials include silicon-containing materials such as silica and polysilicic acid materials, borosilicates, silicates, inorganic glasses, organic polymers such as poly(meth)acrylates, polyurethanes, polystyrene, agarose, polysaccharides such as cellulose, metal oxides such as aluminum oxide, magnesium oxide, titanium oxide, and zirconium oxide, metals such as gold or platinum, Sephadex, Sepharose, polyacrylamide, divinylbenzene polymers, styrene divinylbenzene polymers, dextran, and derivatives thereof; glass or silica. In embodiments, the particles are composed of or contain mineral or polymeric materials such as silica, glass, quartz, polyethylene, polypropylene, polyvinylidene fluoride, polyacrylonitrile, polyvinyl chloride, polyacrylade, methacrylate, or methyl methacrylate.

[0069] The solid phase used in the method of the present invention may be present as a filter in a spin column, but is preferably a magnetic material, such as a paramagnetic, ferrimagnetic, ferromagnetic, or superparamagnetic material. The solid phase of the present invention is not particularly limited to a specific form and may be, for example, in the form of particles (including magnetic particles) or beads (including magnetic beads). Most preferably, the particles are paramagnetic beads or particles. The particles may contain functional groups on their surface for adding protonatable groups, such as Si-O-Si, Si-OH, alcohols, diols, or polyols, carboxylates, amines, phosphates, or phosphonates. Preferably, the magnetic material is completely encapsulated by, for example, silica, polysilicic acid, glass, or a polymer material. In certain preferred embodiments, the solid phase is a silicon-containing particle, preferably a polysilicic acid particle, preferably a magnetic polysilicic acid particle, more preferably a paramagnetic carboxylated silica particle, or a silica-coated paramagnetic particle.

[0070] Preferred paramagnetic particles include silica-coated particles, carboxylated silica particles, carboxylated particles, and particles whose surfaces are modified with cellulose. Examples of carboxylated silica particles include sbeadex® particle suspension (LGC Biosearch Technologies Ltd.). Examples of particles coated with silica alone include MagAttract Suspension A (Qiagen GmbH), MagMAX™ DNA / RNA Binding Beads (ThermoFisher Scientific), mag particle suspension maxi (LGC Biosearch Technologies Ltd.), and MSDS_MagSi-Alpha Beads (magnetic silica microspheres, 1-50 μm, 1%-5% (w / v) in water, Alpha Nanotech Inc.). Examples of carboxylated polystyrene particles include Sera-Mag™ SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva) and Agencourt® AMPure® XP (Beckman Coulter Genomics Inc.). The functional group may be attached directly to the surface of the solid phase or may be part of it. The functional group may also be attached to the solid phase using a suitable spacer or linker, several of which are known in the state of the art and include, for example, hydrocarbon chains, polyethylene, polyglycol, or functionalized silanes, including linear or branched molecules.

[0071] The method according to the invention allows for working with various types of negatively charged surfaces, such as silica surfaces with silanol groups and carboxylated surfaces, or combinations thereof. The choice of surface further expands the degree to which the chemistry can be tailored to a particular sample type.

[0072] Preferably, the particles are made of a material containing reactive alcohol or carboxyl groups, preferably paramagnetic carboxylated silica particles or silica-coated paramagnetic particles, or are provided in a spin column or any other flow-through device. More preferably, they are paramagnetic carboxylated silica particles. The binding mixture has a pH that allows the binding of nucleic acids to the solid phase. The pH value for the binding step is in the range of about 3 to 6, more preferably in the range selected from 4 to 5.5.

[0073] A suitable compound iv) may be an acidifying compound that can be added to the binding solution to establish a pH value of 6 or less. Suitable examples of acidifying compounds include, but are not limited to, acids, acidic buffering agents, such as carboxylic acids, e.g., acetic acid, sodium acetate / acetic acid buffer, citric acid / citrate buffer, maleic acid, malonic acid, tartaric acid, HCl, HClO, HClO, formic acid, boric acid, HSO, HSO, acidic phosphoric acid / phosphate buffer system, MES, or other water-soluble inorganic or organic acids. Acetic acid is preferred. The acidifying compound may also be included in a lysis buffer that is contacted with the sample to establish binding conditions in the binding mixture.

[0074] The binding solution may contain a buffer as compound iv). Suitable buffers include, but are not limited to, carboxylic acid-based buffers, phosphate-based buffers, phosphate buffers, and amino acid-based buffers, such as glycine, glutamate / glutamine, aspartate / asparagine, e.g., MES, MEPES, TRIS, Bis-TRIS, phosphate, borate, or carbonate. The buffer may be included in the lysis buffer and / or binding buffer.

[0075] According to one embodiment, the binding mixture is prepared by contacting a lysis solution with the solid phase and a binding solution. The composition may optionally include an acidifying compound or buffer. Components of the lysis solution, including the lysis buffer, are introduced into the binding mixture.

[0076] The sample, lysis buffer, solid phase, and binding solution may be contacted or added in any order to prepare the final binding mixture. As discussed herein, the sample may be lysed in the lysis buffer before mixing with the binding solution, or the sample may be lysed by the binding solution.

[0077] It has been found that combinations of several binding mechanisms do not compete or interfere with each other, but rather act synergistically and retain the advantages offered by single binding mechanisms while avoiding the disadvantages of each binding mechanism.

[0078] In step (b), the particles with bound nucleic acids are separated from the remaining binding mixture, thereby recovering the particles with bound nucleic acids. Any technique known in the art can be used for this purpose. Suitable techniques include, but are not limited to, magnetic separation when magnetic particles are used, and centrifugation, sedimentation, vacuum application, filtration, etc. when non-magnetic particles are used.

[0079] After step (b), the solid phase may optionally be washed with pure water or an aqueous solution, preferably pure water, as a washing buffer (step c). The aqueous solution may contain a water-miscible organic solvent, such as an alcohol. In particular, the aqueous solution contains about 10% to 80% (v / v) alcohol, preferably about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% alcohol, preferably ethanol or isopropanol. In one embodiment, the aqueous solution contains 70% ethanol. When the aqueous solution contains alcohol, the solid phase may be dried for 1 to 5 minutes. In a preferred embodiment, pure water at a pH of 7 or less is used for the washing step.

[0080] The wash buffer may also contain substances to improve its washing capacity, such as anionic, cationic, or nonionic or zwitterionic detergents, chaotropic salts, buffer substances, or additional ligand-binding compounds.

[0081] The concentration of alcohol in the wash buffer is therefore preferably 5% (v / v) or less, more preferably 3% (v / v) or less, even more preferably 1% (v / v) or less, more preferably 0.5% or less, and most preferably 0% (v / v).

[0082] According to the present invention, only the use of the binding mixture according to the present invention allows the use of pure water as a washing buffer after the binding reaction. In addition, surprisingly, the combination of binding mechanisms also offers the advantage of washing with pure water (pH 7 or less, preferably less than 7) after the binding step. This eliminates the need for additional washing buffers or drying steps to remove all impurities introduced by the binding mixture and sample, prevent alcohol carryover into the eluate, and saves time and chemicals, reduces waste, and results in a very simple protocol.

[0083] According to one embodiment, the wash buffer may contain pure water. Preferably, the wash buffer does not contain organic solvents.

[0084] The method does not require chaotropic salts and is therefore more environmentally friendly as the alcohols and surfactants used can be selected to be environmentally friendly and biodegradable.

[0085] The step of eluting the nucleic acids from the solid phase according to step d) may be carried out at room temperature at a pH in the range of 7.5 to 10, preferably 8 to 10, most preferably 9 to 10. This step is optional but preferred.

[0086] Preferably, an elution solution is added to the particles to which nucleic acids are bound. The elution solution optionally contains a buffer, such as, but not limited to, AMP (2-amino-2-methyl-1-propanol), TRIS, Bis-TRIS, MES, CHAPS, or HEPES. Preferably, AMP, ASMP, or TRIS is used as the buffer agent contained in the elution solution.

[0087] The method according to the present invention does not necessarily contain any chaotropic salts and does not require washing buffers containing detergents, chaotropic salts or organics, and is therefore more environmentally friendly, since the alcohols and detergents used are environmentally compatible.

[0088] Preferably, the kit according to the second aspect is used to carry out the method according to the first aspect. Regarding the properties of this kit, reference is made to the disclosure below.

[0089] According to a second aspect, there is provided a kit for carrying out the method according to the first aspect, the kit comprising: a) lysis buffer, and b) an aqueous binding solution, i) at least one non-ionic surfactant at a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1~4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the composition has a pH selected from 6 or less, 5.5 or less, 4 or less, or 3. an aqueous binding solution, (b) a solid phase containing carboxyl and / or hydroxyl groups capable of selectively binding nucleic acids in the presence of the ligand compound; and (c) optionally, a wash buffer, and (d) one or more elution solutions.

[0090] This kit can be used to carry out the method according to the first aspect. The advantages are as described above. The isolated nucleic acids are of high quality and purity. This makes the method more reliable, which is an important advantage when isolating extracellular nucleic acids, especially for the fields of AgBio, medicine, and / or diagnostics.

[0091] The kit includes a binding solution b). For details regarding the binding solution, please refer to the disclosures described above, which are also applicable here. As discussed above, the binding solution according to the present invention includes i) at least one non-ionic surfactant at a concentration selected from 5% (w / v) to 70% (w / v), 8% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v). According to one embodiment, the kit further includes ii) ethanol or isopropanol.

[0092] According to one embodiment, the kit further comprises iii) a ligand compound comprising at least one protonatable group, which may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine. Preferably, the concentration of diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine is in the range of 0.01 to 100 mM, preferably in the range of 0.1 to 60 mM, more preferably in the range of 0.2 to 50 mM, and even more preferably in the range of 1 to 40 mM.

[0093] In one embodiment, the binding solution b) of the kit according to the invention may be chaotropic salt-free and divalent cation-free.

[0094] The kit further comprises a solid phase comprising carboxyl and / or hydroxyl groups. For details regarding the solid phase, please refer to the disclosures detailed above, which also apply here. As discussed above, the particles are magnetic, preferably paramagnetic.

[0095] According to one embodiment, the kit comprises paramagnetic carboxylated silica particles, for example, sbeadex® particle suspension (LGC Biosearch Technologies Ltd.).

[0096] The kit may further comprise another wash solution, as described above in detail and the disclosure of which also applies here.

[0097] The kit may also include one or more elution solutions, the disclosure of which is described in detail above and which also applies here.

[0098] According to a third aspect, there is provided a formulation for use in a method according to the first aspect, the formulation comprising: i) at least one non-ionic surfactant in concentrations of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v); ii) ethanol or isopropanol at a concentration of 50% to 75%; and iii) a ligand compound comprising at least one protonatable group, the ligand being selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or a corresponding salt thereof; and iv) Acidifying and / or buffering compounds to adjust the pH value of the formulation to between 3 and 6.

[0099] In one embodiment, the formulations according to the present invention may be chaotropic salt-free and divalent cation-free.

[0100] For details regarding the binding solution, please refer to the disclosures set forth above, which are also applicable here. [Example]

[0101] Unless otherwise stated, magnetic sbeadex® particle suspension (LGC Biosearch Technologies Ltd.) was used as the solid phase.

[0102] Example 1: Binding of Nucleic Acids to Carboxylated Silica Solid Supports Using the Nonionic Surfactant Tween 20 A lysate of ground corn seeds was prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS and split into two samples for each reaction.

[0103] Binding was tested using 100 μl of lysate by adding 100 μl of binding solution consisting of water and increasing concentrations of Tween 20 or 150 mM sodium acetate (NaAc) (pH 4.5) and increasing concentrations of Tween 20, and adding 10 μl of sbeadex® particle suspension (LGC Biosearch Technologies Ltd.) (carboxylated silica particles).

[0104] The mixture was vortexed for 2 minutes, the beads were pulled to the side, the supernatant was collected, and the pH was measured. The beads were then washed with 200 μl of water for 30 seconds with constant shaking, and then eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at 60°C.

[0105] The concentration of DNA was quantified via NanoDrop® and Qubit™.

[0106] The purity and quantity of DNA were assessed using a spectrophotometer (Thermo Scientific NanoDrop® Spectrophotometer), which calculates the absorbance of all molecules in the sample that absorb at the wavelength of interest. Nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, so they can contribute to the total absorbance of the sample. Therefore, when using the NanoDrop® Spectrophotometer, it is necessary to purify nucleic acid samples before measurement to ensure the accuracy of the results.

[0107] Qubit™ technology (Thermo Fisher Scientific) is based on a Qubit Fluorometer™, which detects highly specific fluorescent dyes for target molecules of interest in a sample in a Qubit assay. These dyes fluoresce only when bound to their target, even at low concentrations, resulting in highly sensitive measurements. The dyes are absorbed within minutes and can be detected within seconds by the Qubit Fluorometer, which interpolates measurements to a standard curve. In this example, a Qubit Broad Range Kit (Thermo Fisher Scientific) was used.

[0108] Nucleic acids have an absorbance maximum at 260 nm. The ratio of this absorbance maximum to the absorbance at 280 nm is commonly used as an indicator of the purity of both DNA and RNA extracts. For DNA, a 260 / 280 ratio of about 1.8 is generally considered "pure," and for RNA, a ratio of about 2.0 is generally considered "pure."

[0109] As can be seen from Table 1, increasing the concentration of Tween 20 promoted the binding of nucleic acids to silica-coated carboxylated beads (beadex® particle suspension (LGC Biosearch Technologies Ltd.)) at acidic pH. Even more surprising is that the binding was stable, allowing the beads to be washed with pure water, and the DNA was retained on the beads during this wash. Addition of low-salt, basic-buffered water (elution buffer AMP) was sufficient to recover the nucleic acids from the surface. The difference in yield between the Qubit measurement (dsDNA-specific) and the Nanodrop (measurement of DNA and RNA), and the 260 / 280 ratio of approximately 2, indicate that in addition to DNA, RNA must also be co-purified. A 260 / 280 ratio of 1.8 to 2 indicates pure nucleic acid.

[0110] [Table 1]

[0111] In Example 1, we demonstrated that nucleic acids present in a lysate from corn flowers, buffered with Tris (pH 8) using a simple SDS-based lysis buffer with EDTA, could be efficiently purified using LGC sbeadex particles (carboxylated silica particles) as the solid support by adjusting the pH to 4.5 and adding 5% Tween 20 to the final binding mixture, followed by washing the nucleic acids with water for 1 minute and eluting the washed nucleic acids with a low-salt elution buffer (pH 9). Lower DNA yields were already achieved when the mixture had a final Tween 20 content of 2.5% in the binding mixture. Using Tween 20 alone resulted in minimal to no DNA yield, as did simply adjusting the pH to 4.5 with NaAc. In addition to nucleic acid yield, purity also increased with increasing amounts of detergent, likely due to the cleaning properties introduced by the nonionic detergent. This surprisingly demonstrates that the use of a non-ionic detergent under acidic conditions not only transfers nucleic acids to the solid support, but also advantageously introduces detergent properties into the binding mixture, resulting in binding that is strong enough to allow the bound nucleic acids to be washed away with pure water. This binding method has not been described in previous art and can be used without organic solvents, which can be an advantage if the chemistry is to be used in point-of-care testing.

[0112] This embodiment has another significant advantage: in some downstream assays, it is possible to remove the buffer used in the binding reaction and directly elute the nucleic acids from the solid support, bypassing the optional washing step. Surprisingly, the presence of non-ionics is important not only for binding the nucleic acids to the solid support in the presence of high molar salts and / or cationic detergents, but also for keeping the nucleic acids on the beads during the optional washing step with water.

[0113] Example 2: Binding of nucleic acids to carboxylated silica solid supports using Tween 20 at acidic pH A lysate of ground corn seeds is prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and a sample is split into each reaction.

[0114] Binding was tested using 100 μl of lysate by adding 100 μl of binding solution consisting of water and increasing concentrations of Tween 20 or 150 mM NaAc (pH 4.5) in the presence of 60% ethanol, and adding 10 μl of sbeadex® particle suspension (LGC Biosearch Technologies Ltd.) (carboxylated silica particles).

[0115] The mixture was vortexed for 2 minutes, the beads were pulled to the side, the supernatant was collected, and the pH was measured. The beads were then washed with 200 μl of water for 30 seconds with constant shaking, and then eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at 60°C.

[0116] DNA concentrations were quantified via Nanodrop and Qubit (Table 2) and visualized on a 0.8% agarose gel via ethidium bromide and compared to a 1 kb marker.

[0117] The addition of ethanol increased the yield when NaAc (pH 4.5) was not added, likely due to the pH of the binding mixture decreasing to pH 5–6. The difference in yield between Qubit (dsDNA-specific) and Nanodrop (DNA and RNA measurements), and the 260 / 280 ratio of approximately 2, indicate that RNA must be co-purified in addition to DNA. A 260 / 280 ratio of 1.8–2 indicates pure nucleic acid.

[0118] Example 2 demonstrates the use of ethanol in the binding solution. Interestingly, the simultaneous addition of ethanol and Tween 20 resulted in a lower pH value of the binding mixture than the simultaneous addition of water and Tween 20, thus indicating partial purification of nucleic acids from the binding mixture in the presence of ethanol without the addition of acid or additional buffer; thus, other components besides acid or buffer can be used to lower the pH below 6.

[0119] [Table 2]

[0120] Example 3: Binding of nucleic acids to sbeadex beads using non-ionic surfactants at acidic pH A lysate of ground corn seeds was prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and a sample was split into each reaction.

[0121] Binding was tested using 100 μl of lysate by adding 100 μl of 150 mM NaAc (pH 4.5) and 25% of various non-ionic detergents, and 10 μl of LGC Biosearch sbeadex beads.

[0122] The mixture was vortexed for 2 minutes, the beads were pulled to the side, the supernatant was collected, and the pH was measured. The beads were then washed with 200 μl of water for 30 seconds with constant shaking, and then eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at 60°C.

[0123] The concentration of DNA was quantified via nanodrop and qubit, and the pH of the binding mixture was determined using pH strips.

[0124] All nonionic detergents tested, from the Tween, Triton, Tergitol, or Ecosurf groups, were able to promote DNA binding to sbeadex beads, but only the zwitterionic detergent used was unable to promote binding to sbeadex beads (Table 3). Ecosurf® EH-6: 2-Ethylhexanol EO-PO Nonionic Surfactant (Product Number: 64366-70-7) Ecosurf® EH-9: 2-Ethylhexanol EOPO Copolymer (9 EO) (Product Number: 64366-70-7) Ecosurf® SA-7: Ethoxylated Propoxylated Alcohol, Seed Oil Alcohol Ethoxylate 7 EO Ecosurf® SA-9: Ethoxylated Propoxylated Alcohol, Seed Oil Alcohol Ethoxylate 9 EO (Product Number: 68937-66-6) Tergitol® 15-S-7: Secondary alcohol ethoxylate (product number: 84133-50-6) Tergitol® 15-S-9: Secondary alcohol ethoxylate (product number: 84133-50-6) Empigen®: N,N-Dimethyl-N-Dodecylglycine Betaine (Product Number: 66455-29-6)

[0125] [Table 3]

[0126] Example 4: Purification of DNA from various lysis buffers with different properties 1.5 μg of lambda DNA was spiked into 100 μl of LGC Biosearch Technologies Ltd. lysis buffer: LP-PN (CTAB, high salt), LP-PVP (CTAB, high salt), LP-BL (SDS-based, low salt), LP-H (SDS-based, low salt), C1 (GuSCN / TritonX-based), LP-SB (CTAB, GuHCl, Tween-based), 10 mM TE (pH 8), and a 1:1 mixture of LP-SB and LP-PVP.

[0127] The spiked lysis buffer was used to purify DNA by adding 100 μl of 150 mM NaAc (pH 4.5) and 25% Tween 20, and 10 μl of LGC Biosearch Technologies Ltd. Sbeadex beads.

[0128] The mixture was vortexed for 2 minutes, the beads were pulled to the side, the supernatant was collected, and the pH was measured. The beads were then washed with 200 μl of water for 30 seconds with constant shaking, and then eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at 60°C.

[0129] The concentration of DNA was quantified via the nanodrop and qubit.

[0130] The simple addition of a nonionic detergent acidified with NaAc (pH 4.5) enabled the purification of DNA from all the different lysis buffers and even retained the DNA on the surface during water washes. However, purification from CTAB-based lysis buffers (LP-PN and LP-PVP) showed lower yields compared to the other lysis buffers.

[0131] [Table 4]

[0132] Example 5: Binding buffers for several solid phases with different properties Lysates of ground corn seeds and trout biopsy punches were prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and 5 μl proteinase solution, and samples were aliquoted for each reaction. Trout samples were digested for 2 hours at 56°C.

[0133] 100 μl of lysate was used to test binding by adding 100 μl of 150 mM NaAc (pH 4.5) and 25% Tween 20, and 10 μl of the various bead types. A. mag particle suspension maxi (MagMaxi NaCl), LGC Biosearch Technologies Ltd. (silica-coated paramagnetic particles); B. sbeadex® particle suspension (sbeadex beads), LGC Biosearch Technologies Ltd. (silica-coated and carboxylated particles); C. Sera-Mag™ SpeedBead Carboxylate-Modified Magnetic Particles, Cytivia (carboxylated polystyrene beads); D. MagMAX™ DNA / RNA Binding Beads (ThermoFisher Scientific) (silica-coated paramagnetic particles) E. Cellulose-coated paramagnetic beads

[0134] The mixture was vortexed for 2 minutes, the beads were pulled to the side, the supernatant was collected, and the pH was measured. The beads were then washed with 200 μl of water for 30 seconds with constant shaking, and then eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at 60°C.

[0135] The concentration of DNA was quantified via the nanodrop and qubit.

[0136] Paramagnetic beads with various coatings (silica-coated / carboxylated beads (sbeadex beads), silica-only coated beads (LGC magmaxi NaCl and Thermo MagMag DNA / RNA binding beads), and carboxylated polystyrene beads (SpeedBead magnetic carboxylate modified) are all capable of binding DNA at acidic pH and in the presence of the non-ionic detergent Tween 20.

[0137] All solid phases can give good results in terms of yield and purity, suggesting that any surface capable of forming hydrogen bonds can be used.

[0138] Example 6: Effect of pH value Lysates of freeze-dried parsley leaves were prepared using CTAB-based lysis buffer PN (LGC Biosearch Technologies), and lysates of ground corn were prepared using SDS-based lysis buffer BL (LGC Biosearch Technologies), and the samples were divided into separate reactions.

[0139] Binding was tested using 100 μl of the lysate by adding 100 μl of binding buffer consisting of 60% ethanol and 25% Tween 20 and 10 μl of sbeadex® particle suspension (sbeadex beads, LGC Biosearch Technologies Ltd.). The mixture was adjusted to the respective pH value with HCl.

[0140] The mixture was vortexed for 2 minutes, after which the beads were pulled to the side and the supernatant was collected. The beads were then washed with 200 μl of water for 1 minute by constant shaking, pulled to the side, the supernatant was collected, and 50 μl of elution buffer AMP (LGC Biosearch Technologies) was added. The reaction was incubated for 2 minutes at 60°C and vortexed for 30 seconds.

[0141] It has been shown that the optimum exists above pH 3 and below pH 6, preferably between pH 4 and 5.5 (Tables 6A and 6B).

[0142] [Table 5]

[0143] [Table 6]

[0144] Example 7: Selection of organic solvent Lysates of lysed dried parsley leaves were prepared using CTAB-based lysis buffer PN (LGC Biosearch Technologies), and lysates of ground corn were prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and were divided into separate reactions.

[0145] Binding was tested by adding 10 μl of sbeadex® particle suspension (sbeadex beads, LGC Biosearch Technologies Ltd.) to 100 μl of lysate, 100 μl of 60% ethanol, isopropanol, n-propanol, or acetone, and a binding solution consisting of 25% nonionic surfactant Tween 20, and 150 mM NaAc.

[0146] The mixture was vortexed for 2 minutes, after which the beads were pulled to the side and the supernatant was collected. The beads were then washed with 200 μl of water for 1 minute by constant shaking, pulled to the side, the supernatant was collected, and 50 μl of elution buffer AMP (LGC Biosearch Technologies) was added. The reaction was incubated for 2 minutes at 60°C and vortexed for 30 seconds.

[0147] [Table 7]

[0148] Example 8: Various Ligand Compounds Containing at Least One Protonatable Group A lysate of lyophilized parsley leaves was prepared using CTAB-based high-salt lysis buffer PN (LGC Biosearch Technologies) and divided into individual reactions.

[0149] Binding was tested using 100 μl of lysate by adding 10 μl of Sera-Mag™ SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva), SpeedBeads (carboxylated polystyrene beads from GE Healthcare Life Sciences), and 100 μl of 60% ethanol and 25% Tween 20, as well as 150 mM NaAc (pH 4.5), and 10 mM of various amines with different numbers of amino groups ranging from 1 to 5 amines.

[0150] The mixture was vortexed, and after 2 minutes the beads were pulled to the side and the supernatant was collected. The beads were then washed with 400 μl of water for 30 seconds with constant shaking and eluted with 50 μl of elution buffer AMP (LGC Biosearch Technologies) for 2 minutes at room temperature. DNA concentration was quantified via the nanodrop and in part via the qubit.

[0151] [Table 8]

[0152] Example 9: Various solid phases A lysate of ground corn seeds was prepared using SDS-based lysis buffer H (LGC Biosearch Technologies) and divided into individual reactions.

[0153] Using 100 μl of lysate and 100 μl of binding solution consisting of 60% ethanol, 25% Tween 20, and 150 mM NaAc, with or without the addition of 50 mM tetraethylenepentamine, 1.) 10 μl sbeadex® particle suspension (LGC Biosearch Technologies Ltd.) 2.) 5μl LGC mag particle suspension maxi (LGC Biosearch Technologies Ltd.) 3.) 5μl MagAttract Suspension A (Qiagen GmbH) 4.) 10μL MagMAX™ DNA / RNA Binding Beads (ThermoFisher Scientific) 5.) 60 μL Agencourt® AMPure® XP (Beckman Coulter Genomics Inc.) 6.) 40 μL MSDS_MagSi-Alpha Beads (magnetic silica microspheres, 1–50 μm, 1%–5% (w / v) in water, Alpha Nanotech Inc.) 7.) 10μL Sera-Mag™ SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva) Binding was tested by the addition of .

[0154] The mixture was vortexed and after 2 minutes the beads were pulled to the side and the supernatant was collected. A) Wash with 200 μl of water for 30 seconds with constant shaking, or B) Wash with 200 μl of 70% ethanol for 30 seconds with constant shaking and allow to dry for 10 minutes. Subsequently, elution was carried out with 50 μl of elution buffer AMP (LGC Biosearch Technologies) at room temperature for 2 minutes.

[0155] The concentration of DNA was quantified via the nanodrop and in part via the qubit.

[0156] As can be seen from Table 9, when an SDS (anionic surfactant)-based lysis buffer was used for the binding step, washing with water and elution with elution buffer AMP was possible for all test beads. The addition of polyamines showed better purity ratios.

[0157] [Table 9]

[0158] Example 10 100 μl of the lysate was purified using a modified Sbeadex Maxi Plant Kit (1 / 2 Sbeadex Maxi). For short binding, the wash and elution steps were shortened to 5 minutes each, and 10 μl of Sbeadex beads were used to have a similar comparison, eluting with 50 μl of elution buffer AMP.

[0159] Another 100 μl aliquot of the lysate was purified at pH 5 by adding 10 μl of sbeadex beads (from the sbeadex plant kit) and 100 μl of a mixture containing 50% isopropanol, 25% nonionic surfactant, and polyethyleneimine (referred to as Lightning mix). The mixture was vortexed for 10 seconds, and after 1 minute, the beads were pulled to the side using a magnet, and the supernatant was collected. The beads were then washed with 200 μl of water for 1 minute and eluted with 50 μl of elution buffer AMP for 1 minute. DNA purity and quantity were assessed using a Nanodrop™ system. A 260 / 280 value of 1.8–2.0 is indicative of broadly pure, protein-free DNA / RNA.

[0160] The present invention allows for up to 8x faster purification of DNA without sacrificing quality or yield, while reducing liquid waste by up to 50% and requiring 40% fewer consumables.

[0161] [Table 10]

[0162] Example 11 Oak leaf lysates were prepared using CTAB-based lysis buffer PN (from the sbeadex® maxi plant kit) according to the manufacturer's instructions.

[0163] 100 μl of the lysate was used to test the binding by adding 10 μl of sbeadex® beads (sbeadex® maxi plant kit, LGC Biosearch Technologies) and 100 μl of the binding mixture according to Table 11.

[0164] The sample was vortexed for 2 minutes, after which the beads were pulled to the side and the supernatant was collected. The beads were then washed with 400 μl of water for 30 seconds and eluted with 50 μl of TRIS pH = 9 for 2 minutes at room temperature. DNA concentration was quantified via Qubit fluorimeter measurement.

[0165] As can be seen from the results presented in Table 11, by using a cationic surfactant (CTAB)-based lysis buffer, the components of isopropanol, Tween® 20, and triethylenetetramine tetrahydrochloride (TETH) act synergistically to bind nucleic acids and retain them on the bead surface during water washes.

[0166] [Table 11]

[0167] Example 12 A corn seed lysate was prepared using SDS-based lysis buffer H (LGC Biosearch Technologies).

[0168] 100 μl of the lysate was used to test the binding by adding 10 μl of sbeadex® beads (sbeadex® maxi plant kit, LGC Biosearch Technologies) and 100 μl of the binding mixture according to Table 12.

[0169] The sample was vortexed for 2 minutes, after which the beads were pulled to the side and the supernatant was collected. The beads were then washed with 400 μl of water for 30 seconds and eluted with 50 μl of TRIS pH = 9 for 2 minutes at room temperature. DNA concentration was quantified via Qubit fluorimeter measurement.

[0170] As can be seen from the results presented in Table 12, by using an anionic surfactant (SDS)-based lysis buffer, the components of isopropanol, Tween® 20, and triethylenetetramine tetrahydrochloride (TETH) act synergistically to bind nucleic acids and retain them on the bead surface during aqueous washes. The presence of a protic alcohol, while not essential, facilitates binding and enables aqueous washes.

[0171] [Table 12]

[0172] (Comparative Example 13) A 3 mm fish (trout) biopsy punch was lysed overnight at 60°C using 10 μl of proteinase solution (20 mg / ml) in 400 μl of lysis buffer H (SDS-based lysis buffer, manufactured by LGC Biosearch). After lysis, the sample was centrifuged for 1 minute, unlysed material was removed, and 100 μl of the clarified lysate was transferred to two new tubes, 100 μl each.

[0173] To the first tube (sample 1), 100 μl of binding solution (25% Tween 20, 150 mM NaAc (pH 4.5), 70% ethanol) and 10 μl of sbeadex particles (LGC Biosearch) were added.

[0174] To the other tube (sample 2, comparative example) 300 μl of binding buffer (2 M GuHCl, 10% lauryl sarcosine, 30% ethanol, BisTris pH 6.5, (as disclosed in DE 102007009347 A1)) and 10 μl of sbeadex particles (LGC Biosearch) were added.

[0175] The tube was vortexed for 30 seconds, then waited 30 seconds. The beads were separated on a magnet for approximately 50 seconds, and the supernatant was collected. 200 μl of water was added, the tube was vortexed for 30 seconds, and then waited 30 seconds. The beads were separated on a magnet for approximately 10 seconds, and the supernatant was collected. 50 μl of elution buffer was added, the tube was vortexed for 30 seconds, and then incubated at 60° C. for 60 seconds.

[0176] The beads were separated using a magnet and the DNA was analyzed via UV Vis in a Nanodrop (A), and 10 μl of DNA was visualized with SYBR green on a 0.8% agarose gel together with 100 ng lambda marker (Thermo) (50 kb DNA) and NEB 1 kb DNA ladder (B).

[0177] The binding formulation disclosed in DE 102007009347 A1 does not allow for the purification of useful amounts of DNA by water washing.

[0178] [Table 13] * Comparative Example

Claims

1. 1. A method for isolating and / or purifying nucleic acids from a sample, comprising the steps of: a) providing a sample containing nucleic acids and binding said nucleic acids to a solid phase containing carboxyl and / or hydroxyl groups by means of a binding solution, The binding solution i) at least one non-ionic surfactant at a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1～4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the binding mixture, comprising the binding solution and the sample containing the nucleic acid, has a pH between 3 and 6 that allows the nucleic acid to bind to the solid phase; b) separating the solid phase with bound nucleic acids from the binding mixture; c) optionally washing the nucleic acid; and d) eluting the nucleic acid from the solid phase.

2. 10. The method of claim 1, wherein the pH for the binding step is in the range of 4 to 5.

3. The method of claim 1 or 2, wherein the nucleic acid-containing sample comprises a lysis buffer.

4. 4. The method according to claim 1, wherein the binding buffer contains at least one non-ionic detergent in a concentration selected from: i) 5% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v).

5. 5. The method according to claim 1, wherein the binding mixture comprises i) a polysorbate or an alcohol ethoxylate in a concentration of between 20% (w / v) and 30% (w / v).

6. 6. The method according to claim 1, wherein the binding mixture comprises ii) methanol, ethanol, n-propanol, isopropanol, or acetone in a concentration of 15 to 90% (v / v).

7. The method according to one or more of claims 1 to 6, wherein the binding mixture comprises ethanol or isopropanol at a concentration selected from 50% to 75% (v / v).

8. 8. The method according to claim 1, wherein the binding mixture comprises iii) a primary, secondary, tertiary, or quaternary monoamine or polyamine, or an imine having at least one amino group.

9. 9. The method according to claim 1, wherein the solid phase is selected from silica-coated particles, carboxylated silica particles, carboxylated particles, or particles whose surface is modified with cellulose.

10. The method according to one or more of claims 1 to 9, wherein the solid phase comprises paramagnetic carboxylated silica particles or silica-coated paramagnetic particles.

11. 11. The method according to one or more of claims 1 to 10, wherein washing the nucleic acids according to step c) is carried out using pure water or an aqueous solution as a washing buffer.

12. 12. The method according to claim 1, wherein washing the nucleic acids according to step c) is carried out using pure water.

13. 13. The method according to one or more of the preceding claims, wherein eluting the nucleic acid from the solid phase according to step d) is carried out at a pH in the range of 7.5 to 10.

14. A kit for carrying out the method according to one or more of claims 1 to 13, comprising: a) lysis buffer, and b) a binding solution, i) at least one non-ionic surfactant at a concentration of at least 5% (w / v); ii) Optionally, linear or branched C 1～4 an aqueous solution of alcohol or acetone, and iii) a ligand compound optionally containing at least one protonatable group; wherein the binding buffer has a pH selected from 6 or less, 5.5 or less, 4 or less, and 3 or more. Binding solution, c) a solid phase containing carboxyl and / or hydroxyl groups capable of selectively binding nucleic acids in the presence of said ligand compound; and d) optionally one or more cleaning solutions, and e) Optionally, one or more elution solutions.

15. A formulation for use in the method according to one or more of claims 1 to 13, comprising: i) at least one non-ionic surfactant in concentrations of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v); ii) ethanol or isopropanol at a concentration of 50% to 75%; and iii) a ligand compound comprising at least one protonatable group, said ligand being selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine, or triethylamine, or a corresponding salt thereof; and iv) Acidifying and / or buffering compounds to adjust the pH value of the formulation to between 3 and 6.

16. 16. Use of the formulation according to claim 15 in a method for isolating and / or purifying nucleic acids from an aqueous liquid.

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