Method for isolating and / or purifying nucleic acids

Abstract

The present disclosure relates to a method for isolating and / or purifying nucleic acids from a sample, a kit for carrying out the method for isolating and / or purifying nucleic acids, and a formulation for use in the method for isolating and / or purifying nucleic acids.

Description

Technical Field

[0001] The present invention relates to a method for separating and / or purifying nucleic acids from a sample, a kit for carrying out the method for separating and / or purifying nucleic acids, and a preparation for the method for separating and / or purifying nucleic acids. Background Art

[0002] The analysis of nucleic acids (including ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)) is very important in biology, AgBio (agricultural biotechnology), medicine and pharmacology research and diagnostics. For example, by examining the nucleic acid of a cell, the genetic origin and functional activity of the cell can be determined. In addition, genetic markers for detecting and / or predicting disease or phenotypic traits can be identified. In addition, the analysis of RNA and DNA can also be used to identify pathogenic bacteria, fungi and viruses. An important non-limiting example is pathogen nucleic acid (such as viral nucleic acid). Therefore, extracellular nucleic acids are particularly useful in non-invasive diagnosis and prognosis, and can, for example, be used as diagnostic markers in many application areas (such as non-invasive prenatal genetic testing, oncology, transplant medicine or many other diseases), and therefore have diagnostic relevance.

[0003] However, most of these methods for analyzing nucleic acids require nucleic acids as substrates, which have essentially been separated and purified in a previous step (e.g., in a lysis step) from any other cellular components and other contaminants entrained in the sample. Therefore, there is a need for high-throughput methods for separating and / or purifying nucleic acids, which can preferably do so quickly and reliably in an environmentally friendly manner.

[0004] Various methods are known for isolating nucleic acids (such as DNA and RNA) from aqueous samples containing said nucleic acids (eg, biological samples lysed in an aqueous lysis buffer).

[0005] In the fields of molecular diagnostics and modern genetic breeding methods, the steadily increasing number of samples leads to bottlenecks in the workflow, because the assays for nucleic acid analysis or the isolation of nucleic acids to a sufficient purity for analysis are too slow or too costly.

[0006] Typically, nucleic acid extraction ("crude lysis") (e.g., HotShot or (LGC Biosearch Technologies Ltd.), which allows for the rapid release of nucleic acids from the sample material and their direct use for subsequent analysis. A disadvantage of this approach is that, instead of removing the assay inhibitors, they have to be diluted, which reduces the amount of nucleic acid available for the assay (lower concentration). Furthermore, the nucleic acids tend to be degraded due to the harsh processing conditions. Furthermore, different sample types require different methods, which cannot be simplified, so more work is required to find the correct dilution and conditions for each sample type. Furthermore, the error rate in the assay is quite high. With the advent of more expensive downstream assays, such as next-generation sequencing technologies, these crude methods are often no longer acceptable.

[0007] Proper purification of nucleic acids is often slow because the reagents used to lyse the sample and bind the nucleic acids to the solid support must be washed out and removed before downstream analysis, as these reagents can inhibit downstream assays. Typically, nucleic acid purification using solid supports follows a sequence of lysis, binding, two or three wash steps, drying, and elution. As a result, kits typically use large amounts of plastic in processing, generate large amounts of environmentally harmful waste, and are expensive due to the large amounts of chemicals required for purification. Furthermore, each step increases the time it takes to produce results from a sample.

[0008] Several approaches have attempted to overcome this problem. For example, US 2001 / 0018513 A1 involves the use of a solid support comprising a plurality of positively ionizable groups immobilized on a solid phase, which can bind nucleic acids at a pH where the ionizable groups are positively charged, washing the nucleic acids with an aqueous buffer having a pH of 7 or lower, and releasing the nucleic acids at a pH where the ionizable groups are negatively charged, neutral, or less positively charged.

[0009] US2013 / 0158247A1 discloses the use of a positively charged binding ligand containing at least one basic moiety and / or a quaternary ammonium group at a first pH to bind negatively charged nucleic acids before or upon contacting them with a solid support carrying an acidic moiety (such as a carboxyl group, a sulfonic acid group, a phosphonic acid group or an acidic hydroxyl group), washing with an aqueous low-salt buffer, and releasing the nucleic acids at a second pH lower than the first pH and / or with an elution buffer (pH>7 if the ligand contains a quaternary ammonium group).

[0010] CN 113584019 A discloses a reagent and kit for rapidly extracting nucleic acids from FFPE samples, as well as applications of the reagent and kit. The binding buffer contains 1-5 M guanidine, Tween 20 (1-2% v / v), Triton X-100 (1-5% v / v), EDTA (10 to 40 mM), and Tris-HCl (20 to 60 mM) at a pH between 8.0 and 9.5 to produce a binding mixture for absorbing nucleic acids. Reagents for rapidly extracting nucleic acids from FFPE samples also include magnetic beads. However, this method must follow a completely different principle, as it operates at an alkaline pH, requires guanidine salts to absorb nucleic acids on a solid support, and has the disadvantage of requiring washing with an organic solvent.

[0011] DE 102007009 347A1 discloses a method for adsorbing nucleic acids onto polar, weakly acidic surfaces using alcohol, chaotropic salts, and ionic detergents or mixtures thereof, followed by washing the polarly bound nucleic acids with an aqueous buffer solution containing a mediator having primary, secondary, or tertiary amine groups. The positively charged mediator indirectly binds the negatively charged nucleic acids to a solid support and releases the nucleic acids at a pH of 8 or higher. The disadvantage is that the bound material requires a washing step before applying the mediator, and each washing step is time-consuming (5-10 minutes per washing step).

[0012] A disadvantage 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, for example SDS (sodium dodecyl sulfate) or CTAB (cetyltrimethylammonium bromide) for lysis or guanidine salts for binding or keeping the proteins in solution. These usually have to be precipitated before purification, or they have to be washed out of the sample, which means that additional time, effort and cost are required. In addition, the methods described in the prior art still require a large number of washing steps, because the inhibitors are also bound to the solid support, especially when trying to isolate nucleic acids from sample material that contains a large amount of secondary metabolites and other potential PCR inhibitors, such as plant sample material.

[0013] Therefore, there is still a need for a very rapid and environmentally friendly DNA isolation method that can yield high-quality DNA.

[0014] The object of the present invention is to provide a method for isolating and / or purifying nucleic acids from a biological sample containing nucleic acids, which method avoids at least one of the above-mentioned disadvantages of the prior art. In a specific embodiment, an object of the present invention is to provide a simple and rapid method for isolating and / or purifying nucleic acids, which method is suitable for automation and allows the processing of a large number of samples. Summary of the Invention

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

[0016] The present invention relates to a new technology in which a specific binding solution is used to separate and / or purify nucleic acids. Therefore, the method according to the present invention is not based on the known mechanism of binding nucleic acids to a solid phase, in which a combination of alcohol and divalent salts is usually used to bind DNA to a solid phase or to bind negatively charged nucleic acids to a positively charged surface (charge conversion). Similarly, the method according to the present invention does not require chaotropic ("boom" chemistry) salts or kosmotropic salts to promote the binding of nucleic acids to solid phase silica surfaces. The present invention is particularly based on the surprising discovery that the use of a binding solution based on a non-ionic detergent and optionally (but preferably) in combination with a protic alcohol on the solid phase in the method according to the present invention allows for rapid and environmentally friendly purification of nucleic acids without reducing the quality or yield of the nucleic acids. The method according to the present invention allows the separation and / or purification of nucleic acids without being affected by the detergent used in the lysis process. Therefore, the method according to the present invention can be applied to almost all sample types and provides high flexibility.

[0017] According to a first aspect, the present invention provides a method for isolating and / or purifying nucleic acids from a sample, comprising:

[0018] a) providing a sample containing nucleic acid, and binding the nucleic acid to a solid phase containing carboxyl groups and / or hydroxyl groups using a binding solution, wherein the binding solution comprises:

[0019] i) at least one non-ionic detergent at a concentration of at least 5% (w / v);

[0020] ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and

[0021] iii) an optional ligand compound comprising at least one protonatable group,

[0022] wherein the binding mixture comprising the binding solution and the sample containing the nucleic acid has a pH between 3 and 6 to enable binding of the nucleic acid to the solid phase;

[0023] b) separating the solid phase bound to the nucleic acid from the binding mixture;

[0024] c) optionally washing the nucleic acid; and

[0025] d) Elution of nucleic acids from the solid phase.

[0026] According to a second aspect, there is provided a kit for implementing the method according to the first aspect, the kit comprising:

[0027] a) lysis buffer; and

[0028] b) a binding solution comprising:

[0029] i) at least one non-ionic detergent at a concentration of at least 5% (w / v);

[0030] ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and

[0031] iii) an optional ligand compound comprising at least one protonatable group,

[0032] wherein the binding buffer has a pH selected from the group consisting of ≤6, ≤5.5, ≤4, and ≥3;

[0033] c) a solid phase comprising carboxyl and / or hydroxyl groups, the solid phase being capable of selectively binding nucleic acids in the presence of a ligand compound;

[0034] d) optionally one or more washing solutions; and

[0035] e) one or more elution solutions.

[0036] According to one embodiment, the binding solution b) does not contain chaotropic salts and divalent cations. Thus, the binding solution b) of the kit according to the invention does not use any chaotropic salts or divalent cations.

[0037] Yet another aspect of the present disclosure relates to a formulation for use in the method according to the first aspect, comprising:

[0038] i) at least one non-ionic detergent at a concentration of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v);

[0039] ii) 50% to 75% ethanol or isopropyl alcohol;

[0040] 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 their corresponding salts; and

[0041] iv) acidifying and / or buffering compounds for adjusting the pH of the formulation to between 3 and 6.

[0042] According to one embodiment, the preparation according to the invention is free of chaotropic salts and divalent cations.

[0043] Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and the appended claims. However, it should be understood that while the following description, appended claims, and specific examples describe preferred embodiments of the present application, they are presented by way of illustration only. Various changes and modifications within the scope of the disclosed invention will become apparent to those skilled in the art upon reading the following. DETAILED DESCRIPTION

[0044] The present disclosure may be implemented in various forms and should not be construed as being limited to the embodiments shown herein. Instead, these embodiments are provided as examples so that the present disclosure will be comprehensive and complete and fully convey the various aspects and features of the present disclosure to those skilled in the art.

[0045] As used herein, the term "and / or" includes any and all combinations of one or more of the listed associated items. In addition, when describing embodiments of the present disclosure, the use of "may" refers to "one or more embodiments of the present disclosure." In the following description of embodiments of the present disclosure, terms in the singular may include plural forms unless the context clearly indicates otherwise.

[0046] As used herein, the terms "substantially," "approximately," and similar terms are used as terms of approximation, rather than terms of degree, and are intended to account for the inherent variation in measured or calculated values ​​that one of ordinary skill in the art would recognize. Additionally, if the term "substantially" is used in conjunction with a feature that can be expressed in a numerical value, the term "substantially" means a range of + / - 5% of that value centered around that value.

[0047] It should also be understood that the terms "includes," "comprising," "it includes," or "it contains" specify properties, regions, fixed quantities, steps, processes, elements, components, and combinations thereof, but do not exclude other properties, regions, fixed quantities, steps, processes, elements, components, and combinations thereof.

[0048] As used herein, the term "solution" refers in particular to a liquid composition, preferably an aqueous composition. It 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, for example, precipitates, in particular precipitates of contained chemical substances such as stabilizers.

[0049] As used herein, the term "binding mixture" refers to a composition comprising a sample containing nucleic acids and a binding solution that is prepared for the binding step and allows the nucleic acids contained in the sample to bind to the solid phase. Thus, the binding mixture provides suitable conditions for binding of the nucleic acids to the solid phase.

[0050] General Concepts

[0051] The present invention provides a method for isolating and / or purifying nucleic acids from a sample, comprising:

[0052] a) providing a sample containing nucleic acid, and binding the nucleic acid to a solid phase containing carboxyl and / or hydroxyl groups by an aqueous binding solution, wherein the binding solution comprises:

[0053] i) at least one non-ionic detergent at a concentration of at least 5% (w / v);

[0054] ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and

[0055] iii) an optional ligand compound comprising at least one protonatable group,

[0056] wherein the binding mixture comprising the binding solution and the sample containing the nucleic acid has a pH between 3 and 6 to enable binding of the nucleic acid to the solid phase;

[0057] b) separating the solid phase bound to the nucleic acid from the binding mixture;

[0058] c) optionally washing the nucleic acid; and

[0059] d) Elution of nucleic acids from the solid phase.

[0060] The method according to the present invention offers the significant advantage that it is independent of the components of the lysis buffer system used and is therefore not negatively affected by anionic or cationic detergents, chaotropic salts, or high molar concentrations of kosmotropic salts. Consequently, the method according to the present invention can be applied to virtually all types of lysed samples, thus offering a high degree of flexibility.

[0061] The separation and / or purification method according to the present invention has significant advantages over the methods used in the prior art because the method is fast, allows for simple processing of large quantities of samples, requires only minimal processing steps, and does not require a wash buffer other than water readily available in any laboratory, provides high recovery rates and high purity extracellular nucleic acids, and is automatable. Therefore, the method according to the present invention can provide consumers with 3 to 8 times faster throughput without the need to change equipment and without sacrificing the quality or yield of nucleic acids. In addition, the method reduces consumables costs, reduces waste volume and toxic waste volume, and reduces transportation prices and required storage space.

[0062] One or more nucleic acids that can be isolated and / or purified using the methods of the present invention include DNA and RNA of microbial (including viral, bacterial, and fungal) origin, human origin, animal origin, or plant origin, in particular genomic DNA (gDNA), plasmid DNA, PCR fragments, cDNA, rRNA, mRNA, miRNA, cfDNA, siRNA, as well as oligonucleotides and modified nucleic acids (such as so-called peptide nucleic acids or locked nucleic acids (PNA or LNA)). In addition, hybrids formed by DNA and RNA can also be purified, but are not limited thereto.

[0063] The sample to be processed by the method of the present invention can represent any sample comprising nucleic acid, and is preferably a biological sample in natural state or processed form. Preferably, the sample can include body fluids (such as blood, serum, sputum, feces, plasma, sperm, cerebrospinal fluid, saliva, etc.), human, animal or plant tissue and tissue culture, human, animal or plant microbial cells and cell culture and human or animal organs or parts thereof (such as, for example, liver, kidney or lung). In addition, the sample can represent a fluid sample (such as wastewater or drinking water, juice or food), but is not limited thereto. In addition, the sample can represent a processed biological sample (such as, for example, human, animal or plant cell lysate, bacterial lysate, paraffin embedded tissue, aqueous solution or buffer solution or gel of the sample).

[0064] If nucleic acid to be separated is present in cellular material, then can preferably before further processing them according to the method for the present invention, first destroy cellular material according to any method (such as for example by mechanical method, chemical method or enzyme lysis method) for releasing nucleic acid from cell known in the prior art.Preferably, cellular material is cracked to release nucleic acid, and known lysis buffer and method (such as contacting with hypotonic solution, protease, chaotropic agent or solvent of ionic and nonionic detergent, salt, tissue is ground, or adding other lytic enzymes to form sample (also referred to as " lysate " in this article) containing nucleic acid) can be used.Lysate is preferably a lysis solution as the organic matter of sample and lysis buffer.

[0065] In one embodiment, the nucleic acid-containing sample comprises a lysis buffer.

[0066] Examples of lysis buffers are buffers based on low salt anionic detergents such as sodium dodecyl sulfate (SDS), lithium dodecyl sulfate or lauryl dodecyl sulfate, ethylenediaminetetraacetic acid (EDTA) or buffers based on high salt cationic detergents such as cetyltrimethylammonium bromide (CTAB) to produce a solution containing nucleic acids (lysate).

[0067] In the binding step a) of the method, a sample containing nucleic acids (preferably a sample contained in a lysis buffer) is contacted with a solid phase and components of a binding mixture having a pH value of 3 to 6 (preferably 4 to 5) to adsorb the nucleic acids to the solid phase. The binding solution comprises: i) at least one non-ionic detergent (preferably an alcohol ethoxylate or polysorbate at a concentration of at least 5% (w / v)); iii) optionally a ligand compound comprising at least one protonatable group; and ii) optionally, but preferably, a linear or branched C 1-5 An aqueous solution of alcohol or acetone (preferably ethanol or isopropanol). Preferably, the binding solution comprises iv) an acidifying and / or buffering compound to adjust the pH value to 3 to 6 (preferably 4 to 5, more preferably 4 to 4.5).

[0068] For example, the lysate can be added to the binding solution, or the binding solution can be added to the lysate, but preferably the lysate is added to the binding solution. The sample can also be lysed directly by a binding solution containing a lysis buffer.

[0069] The binding solution may contain a solid phase, or the solid phase may be added in a separate step. Alternatively, one or more components i), iii), and iv) of the binding solution may already be present in the lysate. The amounts of components i), ii), and iii) given below refer to the binding solution comprising components i), ii), and iii). The amounts of components i), ii), and iii) are given, respectively, with respect to the final binding mixture comprising the lysate and the binding solution.

[0070] An important feature of the present invention is that i) at least one nonionic detergent is added to the binding solution.

[0071] The term "detergent" refers to amphiphilic organic compounds composed of a hydrophobic, nonpolar hydrocarbon portion (the tail) and a hydrophilic, polar head. The ionic nature of the polar head is the basis for the broad classification of detergents; they can be ionic (charged, either ionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups, but a net charge of zero).

[0072] In a preferred embodiment, the detergent comprises i) at least one non-ionic detergent or a combination of two or more detergents of non-ionic nature.

[0073] Preferably, the nonionic detergent is polysorbate.

[0074] "Polysorbates" (sometimes also called Polysorbates refer to a class of amphiphilic nonionic surfactants derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Polysorbates are typically manufactured by ethoxylating sorbitan prior to esterification with fatty acids. The ethoxylation process results in a sorbitan core with a defined number of polyethylene glycol repeating units distributed across the four different attachment sites (hydroxyl groups) of the sorbitan molecule. Therefore, the term "polysorbate" refers to a mixture of chemical substances that vary, inter alia, by the specific distribution of the polyethylene glycol units across the sorbitan hydroxyl groups.

[0075] Polysorbates are generally named according to the following nomenclature. The first digit indicates the major fatty acid esterified (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.

[0076] Thus, in the binding mixture used in the method according to the present 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 ( 20).

[0077] Non-limiting examples of other nonionic detergents that can be used in the binding mixtures provided herein include polyethylene glycol tert-octylphenyl ether (sold as X-100), copolymers of ethylene oxide and propylene oxide (such as (For example, polyethylene glycol trimethyl nonyl ether (sold by Sigma-Aldrich) TMN-6)), alcohol ethoxylates (e.g., 2-ethylhexanol EO-PO (Cas Reg. No.: 64366-70-7; series, such as DowChemicals EH9 or Sigma-Aldrich 15-S-7, 15-S-9), fatty alcohol alkoxylates or ethoxylates (such as secondary alcohol alkoxylates (e.g., Dow Chemicals' LF-45)) or a combination of two or more of the above substances, preferably alcohol ethoxylates (such as EH9, 15-S-7 or 15-S-9).

[0078] In a preferred embodiment, the nonionic detergent is selected from the group consisting of polysorbates (such as polysorbate 20) and alcohol ethoxylates (such as EH-9, EH-6, EH-9, SA-7, SA-9, 15-S-9, 15-S-7 and 15-S-9, preferably EH-9 or 15-S-7, more preferably 15-S-7).

[0079] The amount of nonionic detergent in the binding solution used in the methods of the present invention can vary. For example, the binding mixture can contain a nonionic detergent 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). A concentration selected from 20% (w / v) to 30% (w / v) is particularly suitable.

[0080] In one embodiment, the content of non-ionic detergent in the binding solution is greater than 10% (w / v), more preferably between 15% and 25% (w / v).

[0081] The final concentration of the nonionic detergent 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%, and it is particularly suitable that the final concentration is selected from 10% (w / v) to 15% (w / v). The presence of a nonionic detergent at a pH between 3 and 6 (preferably between 4 and 5.5) introduces washing properties and helps to drive the nucleic acids to the solid phase. The bound nucleic acids can then be optionally washed by a short water wash (pH below 7) and the bound nucleic acids can be eluted from the solid phase using an alkaline buffer solution with a pH of 7.5 to 13.

[0082] The binding solution according to the present invention further comprises: ii) an optional aqueous solution comprising a linear or branched C1-C4 alcohol (e.g., methanol, ethanol, n-propanol, isopropanol) or acetone at a concentration of 0 to 90% (v / v), preferably ethanol or isopropanol, more preferably isopropanol. According to one embodiment, the binding solution according to the present invention further comprises: ii) methanol, ethanol, n-propanol, isopropanol, or acetone (preferably ethanol or isopropanol) at a concentration of 15% to 90% (preferably selected from 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% and 75% (v / v), more preferably 40%, 45%, 50%, 55%, 55%, 60%, 65% and 70% (v / v), most preferably 50%, 55%, 55% and 60%).

[0083] Preferably, the binding solution according to the present 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)).

[0084] The final concentration of the linear or branched C1-C4 alcohol or acetone in the binding 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% and 70% (v / v) (more preferably 10%, 15%, 20%, 25%, 30%, 35% and 40% (v / v), most preferably 20%, 25% and 30% (v / v)).

[0085] According to one embodiment, the binding solution according to the present invention comprises: i) at least one non-ionic detergent in 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 detergent is polysorbate 20 or an alcohol ethoxylate (e.g. EH-9, EH-6, EH-9, SA-7, SA-9, 15-S-9, 15-S-7 and 15-S-9, preferably EH-9 or 15-S-7, more preferably 15-S-7).

[0086] 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).

[0087] It was found that the use of protic alcohols or acetone in the binding solution allowed the addition of 5% to 40% (w / v) non-ionic detergents without increasing viscosity too much (which would affect pipetting), thus facilitating automation. In addition, for lysates containing cationic detergents (such as CTAB, a detergent widely used for plant DNA purification), the addition of alcohol is required to contain the DNA on the beads. The presence of the non-ionic detergent drives the binding of the nucleic acid to the solid support, but also introduces a washing feature to the binding mixture, making a single, shorter water wash step sufficient to remove impurities.

[0088] This embodiment has significant advantages. In some downstream analyses, it is even possible to remove the buffer used in the binding reaction and elute the nucleic acid directly from the solid support, omitting any washing steps. Surprisingly, the presence of a nonionic detergent is important not only for binding the nucleic acid to the solid support in the presence of high molar concentrations of salt and / or cationic detergents, but also for retaining the nucleic acid on the microbeads during the optional water wash step.

[0089] The binding solution used in the method according to the present invention optionally comprises: iii) a ligand compound comprising at least one protonatable group. The protonatable group is preferably a chemical group that is neutral or uncharged at higher pH values ​​and is protonated at lower pH values, thereby becoming positively charged. In particular, the protonatable group is positively charged at the pH at which nucleic acid binding to the particle occurs. Preferably, the ligand compound comprises one or more (preferably two or more, and most preferably three or more) protonatable groups per molecule.

[0090] Examples of suitable protonatable groups are in particular amino groups (such as primary, secondary and tertiary amino groups) and cyclic amines, aromatic amines and heterocyclic amines. The amino group preferably carries an alkyl, alkenyl, alkynyl and / or aromatic substituent, including cyclic substituents and substituents which, together with the nitrogen atom, form a heterocyclic or heteroaromatic ring. The substituent preferably contains 1 to 20 carbon atoms (more preferably 1 to 12, 1 to 8 or 1 to 6 carbon atoms). They may be straight or branched and may contain heteroatoms (such as oxygen, nitrogen, sulfur, silicon and halogen (e.g., fluorine, chlorine and bromine) atoms). Preferably, the substituent contains no more than 6 (preferably no more than 5 or no more than 4) heteroatoms.

[0091] According to one embodiment, the ligand compound comprising at least one protonatable group is a monoamine or polyamines or imines of a primary amine, a secondary amine, a tertiary amine or a quaternary ammonium with at least one (preferably three or more) amino group. Preferably, the ligand compound can comprise at least two primary amines or at least one secondary amine or tertiary amine. The example of suitable ligand compound is ethylenediamine, diethylenetriamine, triethylenetetramine (TETA), tetraethylenepentamine, pentaethylenehexamine, trimethylamine (TMA), triethylamine (TEA), triethylaminoethyl (TEAE), diethanolamine, diethylamine, diisopropylamine, straight or branched polyethyleneimine (PEI), carboxylation or hydroxyalkylation polyethyleneimine, polyetheramine, spermine, spermidine, polyamidoamine (PAMAM) dendrimer, polyallylamine, polyvinylamine, N-ethylmorpholine, polylysine, polyarginine, protamine or tetraazacycloalkane and its corresponding salt.

[0092] According to a preferred embodiment iii), the ligand compound comprising at least one protonatable group can 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 preferred ligand compounds can be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine or triethylamine or their corresponding salts. Particularly preferably, tetraethylenepentamine or its corresponding salt is used.

[0093] Diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine or triethylamine is used in a concentration 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, even more preferably in the range of 1 to 40 mM.

[0094] According to one embodiment, the binding solution according to the present invention comprises: i) at least one non-ionic detergent 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 ligand compound 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, even more preferably in the range of 1 to 40 mM.

[0095] According to one embodiment, the binding solution according to the present invention comprises: i) a non-ionic detergent 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 comprising at least one protonatable group, which ligand compound may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine or triethylamine at a concentration of 0.1 to 40 mM or their corresponding salts; wherein i) the non-ionic detergent is polysorbate 20 or an alcohol ethoxylate (such as EH-9, EH-6, EH-9, SA-7, SA-9, 15-S-9, 15-S-7 and 15-S-9), preferably, the nonionic detergent is an alcohol ethoxylate (such as EH9 or 15-S-7).

[0096] 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 comprising at least one protonatable group, which ligand compound may be selected from diethanolamine, diethylamine, diisopropylamine, ethylenediamine, triethylenetetramine, spermidine, spermine, tetraethylenepentamine or triethylamine or their corresponding salts. 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, even more preferably in the range of 1 to 40 mM.

[0097] In the method according to the invention a nucleic acid binding solid phase is used. In one embodiment of the method according to the invention, the binding mixture and the solid phase comprising carboxyl and / or hydroxyl groups are mixed (preferably for at least 30 seconds, more preferably for 2 minutes) before separation of the solid phase from the binding mixture.

[0098] Examples of solid phase materials may include silicon-containing materials (such as silicon dioxide and polysilicic acid materials, borosilicates, silicates, inorganic glasses), organic polymers (such as polymethacrylates, polyurethanes, polystyrenes), agaroses, polysaccharides (such as cellulose), metal oxides (such as aluminum oxide, magnesium oxide, titanium oxide, and zirconium oxide), metals (such as gold or platinum), cross-linked dextran, agaroses, polyacrylamides, divinylbenzene polymers, styrene-divinylbenzene polymers, dextran and its derivatives; glass or silicon dioxide. In some embodiments, the particles are made of or contain mineral or polymer materials (such as silicon dioxide, glass, quartz, polyethylene, polypropylene, polyvinylidene fluoride, polyacrylonitrile, polyvinyl chloride, polyacrylates, methacrylates, or methyl methacrylates).

[0099] The solid phase used in the method for the present invention can exist as the filter element in the centrifugal column, but preferably can be magnetic, including paramagnetic material, ferrimagnetic material, ferromagnetic material or superparamagnetic material.Solid phase of the present invention is especially not limited to special form, and can be, for example, granular form, including magnetic particles and microbeads (including magnetic beads).Most preferably, particle can be paramagnetic beads or particles.Particle can comprise the functional group for attaching protonated groups, such as Si-O-Si, Si-OH, alcohol, diol or polyol, carboxylate, amine, phosphate or phosphonate at its surface.Preferably, magnetic material is for example fully encapsulated by silicon dioxide, polysilicic acid, glass or polymeric material.In some preferred embodiments, solid phase is silicon-containing particles (preferably polysilicic acid particles, preferably magnetic polysilicic acid particles, more preferably paramagnetic carboxylated silicon dioxide particles or paramagnetic particles coated with silicon dioxide).

[0100] Preferred paramagnetic particles may be silica coated particles, carboxylated silica particles, carboxylated particles or particles whose surface is modified with cellulose. Examples of carboxylated silica particles are Particle suspensions (LGCBiosearch Technologies Ltd.); examples of particles coated only with silica are MagAttract Suspension A (Qiagen GmbH), Mag MAX TMDNA / RNA binding beads (Binding Beads) (ThermoFisher Scientific), magnetic particle suspension (mag particle suspension maxi) (LGCBiosearch Technologies Ltd.) and MSDS_MagSi-Alpha Beads (magnetic silica microspheres, 1-50 μm, 1%-5% (w / v) in water, Alpha Nanotech Inc.); an example of carboxylated polystyrene particles is Sera-Mag TM SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva) and XP (Beckman Coulter Genomics Inc.). The functional group can be attached directly to the surface of the solid phase or can be part of the surface of the solid phase. The functional group can also be attached to the solid phase using an appropriate spacer or linker, many of which are known in the art, including, for example, hydrocarbon chains, polyethylene, polyethylene glycol, or functionalized silanes, including linear or branched molecules.

[0101] The method according to the present invention is applicable to different types of negatively charged surfaces (such as silica surfaces with silanol groups and carboxylated surfaces or a combination thereof). The choice of surface further expands the extent to which the chemistry can be tuned to a certain sample type.

[0102] Preferably, the particles are made of a material containing a reactive alcohol or carboxyl group and are preferably paramagnetic carboxylated silica particles or silica-coated paramagnetic particles, or are arranged in a spin column or any other flow-through device. More preferably, paramagnetic carboxylated silica particles are used. The pH of the binding mixture should allow the nucleic acid to bind to the solid phase. The pH of the binding step is in the range of about 3 to 6 (more preferably in the range selected from 4 to 5.5).

[0103] Suitable compound iv) can be an acidifying compound, which can be added to the binding solution to establish a pH value less than 6. Suitable examples of acidifying compounds include, but are not limited to, acids and acidic buffers (such as carboxylic acids), such as acetic acid, sodium acetate / acetic acid buffer, citric acid / citrate buffer, maleic acid, malonic acid, tartaric acid, HCl, HClO 4 , HClO 3 , formic acid, boric acid, H 2 SO 4 , H 2 SO 3 , acidic phosphoric acid / phosphate buffer system, MES or other water-soluble inorganic or organic acids. Acetic acid is preferred. The acidifying compound can also be included in a lysis buffer that is contacted with the sample to establish binding conditions in the binding mixture.

[0104] The binding solution may include 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, aspartic acid / asparagine), such as MES, MEPES, TRIS, Bis-TRIS, phosphates, borates, or carbonates. The buffer may be included in the lysis buffer and / or the binding buffer.

[0105] According to one embodiment, a binding mixture is prepared by contacting a lysate with a solid phase and a binding solution. The composition may optionally comprise an acidifying compound or a buffer. The lysate components, including the lysis buffer, are added to the binding mixture and are thus present in the binding mixture.

[0106] The sample, lysis buffer, solid phase and binding solution can be contacted or added in any order to prepare the final binding mixture. As described herein, the sample can be lysed in lysis buffer before mixing with the binding solution, or the sample can be lysed by the binding solution.

[0107] It was found that the combination of several binding mechanisms does not compete or interfere with each other, but rather works synergistically and maintains the advantages provided by a single binding mechanism while avoiding the disadvantages of each binding mechanism.

[0108] In step (b), the particles bound to the nucleic acid are separated from the remaining binding mixture. Thus, the particles bound to the nucleic acid are collected. To this end, any method known in the art can be used. Suitable methods include, but are not limited to: magnetic separation (when using magnetic particles); centrifugation (for example, when using non-magnetic particles); precipitation; vacuum extraction; filtration, etc.

[0109] After step (b), the solid phase can be optionally washed with pure water or an aqueous solution as a washing buffer (preferably pure water (step c)). The aqueous solution can contain an organic solvent miscible with water (such as an alcohol), in particular an aqueous solution containing 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. In the case where the aqueous solution contains alcohol, the solid phase can be dried for 1 to 5 minutes. In a preferred embodiment, pure water is used for washing at pH<7.

[0110] The wash buffer may also contain substances that enhance its washing power (such as anionic, cationic or nonionic or zwitterionic detergents, chaotropic salts, buffer substances or additional ligand binding compounds).

[0111] Therefore, the concentration of alcohol in the wash buffer is preferably <5% (v / v) (more preferably <3% (v / v), even more preferably <1% (v / v), more preferably <0.5%, most preferably 0% (v / v)).

[0112] According to the present invention, only by using the binding mixture according to the present invention is it possible to use pure water as a washing buffer after the binding reaction. Moreover, surprisingly, the combination of binding mechanisms also offers the advantage of washing with pure water (pH ≤ 7, preferably < 7) after the binding step. Thus, all impurities introduced by the binding mixture and the sample can be removed, alcohol residues in the elution are prevented, and no additional washing buffer or drying step is required to prevent alcohol residues in the elution, thus saving time and chemical reagents and reducing waste, and resulting in a very simple solution.

[0113] According to one embodiment, the wash buffer may contain pure water. Preferably, the wash buffer does not contain an organic solvent.

[0114] This method does not require chaotropic salts and is therefore more environmentally friendly, as the alcohols and detergents used are environmentally friendly and can be selected to be biodegradable.

[0115] The step of eluting the nucleic acids from the solid phase according to step d) can be performed at room temperature in a pH range of 7.5 to 10, preferably 8 to 10, most preferably 9 to 10. This step is optional, but also preferred.

[0116] Preferably, an elution solution is added to the particles bound to the nucleic acid. The elution solution may optionally contain a buffer (such as AMP (2-amino-2-methyl-1-propanol), TRIS, Bis-TRIS, MES, CHAPS, and HEPES), but is not limited to these buffers. Preferably, AMP, ASMP, or TRIS is used as the buffer contained in the elution solution.

[0117] The method according to the present invention does not necessarily contain any chaotropic salts and does not require a washing buffer containing detergents, chaotropic salts or organic substances, and is therefore more environmentally friendly because the alcohols and detergents used are compatible with the environment.

[0118] Preferably, the method according to the first aspect is performed using a kit according to the second aspect. Regarding the features of the kit, see the subsequent disclosure.

[0119] According to a second aspect, a kit for implementing the method according to the first aspect is provided. The kit comprises:

[0120] a) lysis buffer; and

[0121] b) an aqueous binding solution comprising:

[0122] i) at least one non-ionic detergent at a concentration of at least 5% (w / v);

[0123] ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and

[0124] iii) an optional ligand compound comprising at least one protonatable group, wherein the pH of the composition is selected from ≤6, ≤5.5, and ≤4, or is 3;

[0125] (b) a solid phase comprising carboxyl and / or hydroxyl groups, wherein the solid phase is capable of selectively binding nucleic acids in the presence of a ligand compound;

[0126] (c) optional wash buffer; and

[0127] (d) one or more elution solutions.

[0128] This kit can be used to implement the method according to the first aspect. The advantages are as described above. The isolated nucleic acids are of high quality and high purity. Thus, the method becomes more reliable, which is an important advantage, especially when isolating extracellular nucleic acids for use in agricultural biology (AgBi)o, medicine and / or diagnostics.

[0129] The kit includes a binding solution b). Details regarding the binding solution have been described above; please refer to the above disclosure, which is applicable here. As described above, the binding solution according to the present invention includes: i) at least one non-ionic detergent 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 also includes ii) ethanol or isopropanol.

[0130] According to one embodiment, the kit further comprises: iii) a ligand compound comprising at least one protonatable group, the ligand compound being 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, even more preferably in the range of 1 to 40 mM).

[0131] In one embodiment, the binding solution b) of the kit according to the present invention may be free of chaotropic salts and divalent cations.

[0132] In addition, the kit also includes a solid phase comprising carboxyl and / or hydroxyl groups. Details about the solid phase have been described in detail above, please refer to the above disclosure, which is applicable here. As mentioned above, the particles are magnetic (preferably paramagnetic).

[0133] According to one embodiment, the kit includes paramagnetic carboxylated silica particles (such as Particle suspension (LGC Biosearch Technologies Ltd.).

[0134] In addition, the kit may also include one or more washing solutions. Details are described above, see the above disclosure, which is applicable here.

[0135] The kit may also include one or more elution solutions. Details are described above, see the above disclosure, which is applicable here.

[0136] According to a third aspect, a formulation for use in the method according to the first aspect is provided. The formulation comprises:

[0137] i) at least one non-ionic detergent at a concentration of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v);

[0138] ii) 50% to 75% ethanol or isopropyl alcohol;

[0139] 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 their corresponding salts; and

[0140] iv) acidifying and / or buffering compounds for adjusting the pH of the formulation to between 3 and 6.

[0141] In one embodiment, the formulation according to the present invention may be free of chaotropic salts and divalent cations.

[0142] Details regarding the binding solution have been described above, please refer to the above disclosure, which is applicable here.

[0143] Example

[0144] Unless otherwise stated, magnetic A particle suspension (LGC Biosearch Technologies Ltd.) served as the solid phase.

[0145] Example 1:

[0146] Binding of nucleic acids to carboxylated silica solid supports using the nonionic detergent Tween 20

[0147] Lysates of ground corn seeds were prepared with 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and the lysates were split into two samples for each reaction.

[0148] The cells were then lysed by using 100 μl of lysate, adding 100 μl of binding solution (pH 4.5) consisting of water and increasing concentrations of Tween 20 or 150 mM sodium acetate (NaAc) and increasing concentrations of Tween 20 and adding Particle suspension (LGC Biosearch Technologies Ltd.) (carboxylated silica particles) was used to test binding.

[0149] The mixture was vortexed; after 2 minutes, the beads were pulled aside, the supernatant removed, and the pH measured. The beads were then washed with 200 μl of water for 30 seconds by continuous shaking, followed by elution in 50 μl of elution buffer AMP purchased from LGC Biosearch Technologies Ltd. at 60°C for 2 minutes.

[0150] pass and Qubit TM Quantify DNA concentration.

[0151] In the spectrophotometer (Thermo Scientific The purity and quantity of DNA are assessed on a spectrophotometer (spectrophotometer), which measures the absorbance of all molecules in the sample that absorb at the wavelength of interest. Since nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, they will contribute to the total absorbance of the sample. Therefore, to ensure the use of Spectrophotometer can provide accurate results, and nucleic acid samples need to be purified before measurement.

[0152] Qubit TM Technology (Thermo Fisher Scientific) based on the Qubit fluorometer TMThe fluorometer uses a highly specific fluorescent dye to detect target molecules in the sample in the Qubit assay. These dyes fluoresce only when bound to their target (even at low concentrations), making the measurement very sensitive. The dye is absorbed within minutes and can be read by the Qubit fluorometer within seconds, which interpolates the readings into a standard curve. In this example, the Qubit Broad Spectrum Kit (Thermo Fisher Scientific) was used.

[0153] Nucleic acids have a maximum absorbance at 260 nm. Typically, the ratio of this maximum absorbance to the absorbance at 280 nm is used as a measure of purity in DNA and RNA extractions. For DNA, a 260 / 280 ratio of approximately 1.8 is generally considered "pure," while for RNA, a ratio of approximately 2.0 is generally considered "pure."

[0154] As can be seen from Table 1, increasing the concentration of Tween 20 is beneficial to the binding of nucleic acids to silica-coated carboxylated microbeads ( The beads were then bound to a suspension of particles (LGC Biosearch Technologies Ltd.). Even more surprisingly, the binding was so stable that the beads could be washed with pure water, and the DNA was retained on the beads during the wash process. Adding a low-salt, alkaline buffered water (elution buffer AMP) was sufficient to recover the nucleic acids from the surface. The difference in yield between the Qubit measurement (for dsDNA) and the Nanodrop (measuring DNA and RNA), as well as the 260 / 280 ratio of approximately 2, indicated that RNA must be co-purified in addition to DNA. A 260 / 280 ratio of 1.8 to 2 indicates pure nucleic acid.

[0155] Table 1:

[0156]

[0157]

[0158] In reality ShiIn Example 1, it is shown that nucleic acids present in a lysate derived from cornflower (using a simple SDS-based lysis buffer containing EDTA and buffered with Tris (pH 8)) are efficiently purified by using LGC sbeadex particles (carboxylated silica particles) as a solid phase support, 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). If the final content of Tween 20 in the binding mixture is 2.5%, a lower DNA yield is already obtained. The use of Tween 20 alone and the use of NaAc alone to adjust the pH to 4.5 result in little or no DNA yield. In addition to the yield, the purity of the nucleic acids is also improved with increasing the amount of detergent used, which may be due to the washing properties introduced by the non-ionic detergent. This surprisingly demonstrates that the use of non-ionic detergents under acidic conditions not only drives the nucleic acids to the solid support but also advantageously introduces washing properties into the binding mixture, achieving sufficiently strong binding that the bound nucleic acids can be washed with pure water. This binding method is not described in the prior art and can be applied without organic solvents, which may be an advantage in cases where chemical substances are used in point-of-care testing.

[0159] This embodiment has other significant advantages. In some downstream analyses, it is even possible to remove the buffer used in the binding reaction and elute the nucleic acids directly from the solid support, omitting any washing steps. Surprisingly, the presence of a nonionic detergent is important not only for binding the nucleic acids to the solid support in the presence of high molar concentrations of salt and / or cationic detergents, but also for retaining the nucleic acids on the microbeads during the optional water wash step.

[0160] Example 2:

[0161] Binding of nucleic acids to carboxylated silica solid supports using Tween 20 at acidic pH

[0162] Lysates of ground corn seeds were prepared with 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and samples were allocated for each reaction.

[0163] The cells were then lysed by using 100 μl of lysate, adding 100 μl of binding solution consisting of water and increasing concentrations of Tween 20 or 150 mM NaAc (pH 4.5 in a 60% ethanol background), and adding A particle suspension (LGCBiosearch Technologies Ltd.) (carboxylated silica particles) was used to test binding.

[0164] The mixture was vortexed; after 2 minutes, the beads were pulled aside, the supernatant removed, and the pH measured. The beads were then washed with 200 μl of water for 30 seconds by continuous shaking, followed by elution in 50 μl of elution buffer AMP purchased from LGC Biosearch Technologies Ltd. at 60°C for 2 minutes.

[0165] DNA concentrations were quantified by Nanodrop and Qubit (Table 2) and visualized on 0.8% agarose gels using ethidium bromide and a 1 kb marker.

[0166] When NaAc (pH 4.5) was not added, the addition of ethanol increased the yield, likely due to a decrease in the pH of the binding mixture to 5 to 6. The difference in yield between the Qubit measurement (for dsDNA) and the Nanodrop (which measures both DNA and RNA), as well as the 260 / 280 ratio of approximately 2, suggests that RNA must be copurified in addition to DNA. A 260 / 280 ratio of 1.8 to 2 indicates pure nucleic acid.

[0167] Reality Shi Example 2 demonstrates the use of ethanol in the binding solution. Interestingly, the addition of ethanol and Tween 20 resulted in a lower pH for the binding mixture compared to the addition of water and Tween 20, thus explaining the partial purification of nucleic acids from the binding mixture in the presence of ethanol without the addition of acid or additional buffer. It is also possible to use components other than acid or buffer to lower the pH to below 6.

[0168] Table 2:

[0169]

[0170]

[0171] Example 3:

[0172] Binding of nucleic acids to sbeadex microbeads using non-ionic detergents at acidic pH

[0173] Lysates of ground corn seeds were prepared with 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS, and samples were allocated for each reaction.

[0174] Binding was tested by taking 100 μl of lysate, adding 100 μl of 150 mm NaAc (pH 4.5) and 25% of different non-ionic detergents and adding 10 μl of LGC Biosearch sbeadex beads.

[0175] The mixture was vortexed; after 2 minutes, the beads were pulled aside, the supernatant removed, and the pH measured. The beads were then washed with 200 μl of water for 30 seconds by continuous shaking, followed by elution in 50 μl of elution buffer AMP purchased from LGC Biosearch Technologies Ltd. at 60°C for 2 minutes.

[0176] DNA concentration was quantified by NanoDrop and Qubit. The pH of the binding mixture was measured using pH strips.

[0177] All tested nonionic detergents from the Tween, Triton, Tergitol or Ecosurf families were able to promote the binding of DNA to sbeadex microbeads, while zwitterionic detergents alone did not promote binding to sbeadex microbeads (Table 3).

[0178] EH-6: 2-Ethylhexanol EO-PO nonionic surfactant (CAS No.: 64366-70-7)

[0179] EH-9: 2-Ethylhexanol EOPO copolymer (9EO) (CAS No.: 64366-70-7)

[0180] SA-7: Alcohol Ethoxylate Propoxylate, Seed Oil Alcohol Ethoxylate 7EO

[0181] SA-9: Ethoxypropoxylated alcohol, seed oil alcohol ethoxylate 9EO (CAS No. 68937-66-6)

[0182] 15-S-7: Secondary alcohol ethoxylate (CAS No.: 84133-50-6)

[0183] 15-S-9: Secondary alcohol ethoxylate (CAS No.: 84133-50-6)

[0184] N,N-Dimethyl-N-dodecylglycine-betaine (CAS No.: 66455-29-6)

[0185] Table 3:

[0186]

[0187] Example 4:

[0188] Purify DNA from different lysis buffers with different properties

[0189] 1.5 μg of λ DNA (Lambda DNA) was added to 100 μl of LGC Biosearch Technologies Ltd.'s 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, GuHCI, Tween-based), 10 mM TE (pH 8), and a 1:1 mixture of LP-SB and LP-PVP.

[0190] DNA was purified by spiking lysis buffer, adding 100 μl of 150 mM NaAc (pH 4.5) and 25% Tween 20 and adding 10 μl of sbeadex microbeads from LGC Biosearch Technologies Ltd.

[0191] The mixture was vortexed; after 2 minutes, the beads were pulled aside, the supernatant removed, and the pH measured. The beads were then washed with 200 μl of water for 30 seconds by continuous shaking, followed by elution in 50 μl of elution buffer AMP purchased from LGC Biosearch Technologies Ltd. at 60°C for 2 minutes.

[0192] DNA concentration was quantified by NanoDrop and Qubit.

[0193] The simple addition of a non-ionic detergent acidified with NaAc (pH 4.5) allowed DNA to be purified from all different lysis buffers and additionally retained the DNA on the surface during the water washes. However, purification from CTAB-based lysis buffers (LP-PN and LP-PVP) showed lower yields compared to the other lysis buffers.

[0194] Table 4:

[0195]

[0196]

[0197] Example 5:

[0198] Binding buffers for several solid phases with different properties

[0199] Lysates of ground corn seeds and trout biopsy samples were prepared with 20 mM Tris (pH 8), 20 mM EDTA, 1% SDS and 5 μl of protease solution and samples were allocated for each reaction. Trout samples were digested at 56°C for 2 hours.

[0200] Binding was tested by using 100 μl of lysate, adding 100 μl of 150 mM NaAc (pH 4.5) and 25% Tween 20 and adding 10 μl of different types of microbeads.

[0201] A. Magnetic particle suspension maxi (MagMaxi NaCl), LGC Biosearch Technologies Ltd. (paramagnetic particles coated with silica);

[0202] B. Particle suspension (sbeadex Beads), LGC Biosearch Technologies Ltd. (silica-coated carboxylated particles);

[0203] C.Sera-Mag TM SpeedBead carboxylate-modified magnetic particles (Carboxylate-Modified Magnetic Particles), Cytivia (carboxylated polystyrene microbeads);

[0204] D.MagMAX TM DNA / RNA Binding Beads (ThermoFisher Scientific) (silica-coated paramagnetic particles);

[0205] E. Cellulose-coated paramagnetic microbeads.

[0206] The mixture was vortexed; after 2 minutes, the beads were pulled aside, the supernatant removed, and the pH measured. The beads were then washed with 200 μl of water for 30 seconds by continuous shaking, followed by elution in 50 μl of elution buffer AMP purchased from LGC Biosearch Technologies Ltd. at 60°C for 2 minutes.

[0207] DNA concentration was quantified by NanoDrop and Qubit.

[0208] All of the different coated paramagnetic beads (silica-coated / carboxylated beads (sbeadex Beads), beads coated with silica alone (LGC mag maxi NaCl and Thermo MagMag DNA / RNA binding beads), and carboxylated polystyrene beads (SpeedBead magnetic carboxyl-modified beads) were able to bind DNA at acidic pH in the presence of the non-ionic detergent Tween 20.

[0209] All solid phases provided good results in terms of yield and purity, indicating that any surface capable of hydrogen bonding can be used.

[0210] Example 6:

[0211] Affects pH

[0212] Lysates of freeze-dried parsley leaves were prepared using CTAB-based lysis buffer PN purchased from LGC Biosearch Technologies Ltd., and lysates of ground corn were prepared using SDS-based lysis buffer BL purchased from LGC Biosearch Technologies Ltd., and samples were allocated for each reaction.

[0213] By using 100 μl of lysate and 100 μl of binding buffer composed of 60% ethanol and 25% Tween 20 and adding 10 μl The binding was tested using a particle suspension (sbeadex Beads, LGC Biosearch Technologies Ltd.). The mixture was adjusted to the corresponding pH value with HCl.

[0214] The mixture was vortexed; after 2 minutes, the beads were pulled aside and the supernatant removed. The beads were then washed with 200 μl of water for 1 minute with continuous shaking, the beads were pulled aside, the supernatant removed, and 50 μl of elution buffer AMP (available from LGC Biosearch Technologies Ltd.) was added. The reaction was incubated at 60°C for 2 minutes and vortexed for 30 seconds.

[0215] It was shown that there is an optimum above pH 3 and below pH 6, preferably between pH 4 and 5.5 (Table 6a and Table 6b).

[0216] Table 6a:

[0217]

[0218] Table 6b:

[0219]

[0220] Example 7:

[0221] Selection of organic solvents

[0222] Lysates of freeze-dried parsley leaves were prepared using CTAB-based lysis buffer PN purchased from LGC Biosearch Technologies Ltd., and lysates of ground corn were prepared using 20 mM Tris (pH 8), 20 mM EDTA, and 1% SDS and divided for each reaction.

[0223] The lysate was purified by using 100 μl of lysate, 100 μl of binding solution consisting of 60% ethanol, isopropanol, n-propanol or acetone and 25% non-ionic detergent Tween20 and 150 mM NaAc and adding 10 μl Particle suspensions (sbeadexBeads, LGC Biosearch Technologies Ltd.) were used to test binding.

[0224] The mixture was vortexed; after 2 minutes, the beads were pulled aside and the supernatant removed. The beads were then washed with 200 μl of water for 1 minute with continuous shaking, the beads were pulled aside, the supernatant removed, and 50 μl of elution buffer AMP (available from LGC Biosearch Technologies Ltd.) was added. The reaction was incubated at 60°C for 2 minutes and vortexed for 30 seconds.

[0225] Table 7:

[0226]

[0227] Example 8:

[0228] Different ligand compounds containing at least one protonatable group

[0229] Lysates of freeze-dried parsley leaves were prepared using CTAB-based high salt lysis buffer PN purchased from LGC Biosearch Technologies Ltd. and divided for each reaction.

[0230] By using 100 μl of lysate and adding 10 μl of Sera-Mag TMBinding was tested using SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva), SpeedBeads (purchased from GE Healthcare Life Sciences, carboxylated polystyrene microbeads) and 100 μl of 60% ethanol and 25% Tween 20, as well as 150 mM NaAc (pH 4.5) and 10 mM of different amines with different numbers of amino groups (1 amino group to 5 amino groups).

[0231] The mixture was vortexed; after 2 minutes, the beads were pulled aside and the supernatant removed. The beads were then washed with 400 μl of water for 30 seconds with continuous shaking and eluted in 50 μl of elution buffer AMP from LGC Biosearch Technologies Ltd. for 2 minutes at room temperature. DNA concentration was quantified by NanoDrop and Qubit.

[0232] Table 8:

[0233]

[0234] Example 9:

[0235] Different solid phases

[0236] Lysates of ground corn seeds were prepared using SDS-based lysis buffer H purchased from LGC Biosearch Technologies and divided for each reaction.

[0237] Binding was tested using 100 μl of lysate, 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, and the following:

[0238] 1.) 10 μl Particle suspension (LGC Biosearch Technologies Ltd.);

[0239] 2.) 5 μl LGC mag particle suspension maxi (LGC Biosearch Technologies Ltd.);

[0240] 3.) 5 μl MagAttract Suspension A (Qiagen GmbH);

[0241] 4.) 10μL MagMAX TM DNA / RNA binding beads (ThermoFisherScientific);

[0242] 5.) 60 μL XP (Beckman Coulter Genomics Inc.);

[0243] 6.) 40 μL MSDS_MagSi-Alpha Beads (Magnetic Silica Microspheres, 1-50 μm, 1%-5% (w / v) in water, Alpha Nanotech Inc.);

[0244] 7.) 10 μL Sera-Mag TM SpeedBead Carboxylate-Modified Magnetic Particles (Cytiva)

[0245] The mixture was vortexed; after 2 minutes, the beads were pulled aside and the supernatant was removed.

[0246] A) Wash the beads with 200 μl of water by continuous shaking for 30 seconds or

[0247] B) The beads were washed with 200 μl 70% ethanol for 30 seconds with continuous shaking, dried for 10 minutes, and then eluted in 50 μl elution buffer AMP (available from LGC Biosearch Technologies Ltd.) for 2 minutes at room temperature.

[0248] DNA concentration was quantified by NanoDrop and Qubit.

[0249] As can be seen from Table 9, if the binding process is performed using an SDS (anionic detergent) based lysis buffer, all the beads tested can be washed with water and eluted with the elution buffer AMP. The addition of polyamines showed a better purity ratio.

[0250] Table 9:

[0251]

[0252]

[0253] Example 10:

[0254] 100 μl of lysate was purified using a modified version of the sbeadex maxi plant kit (1 / 2 sbeadex maxi). In short binding, the wash and elution steps were each shortened to 5 minutes, 10 μl of sbeadex beads were used for like comparison, and the beads were eluted in 50 μl of elution buffer AMP.

[0255] Another 100 μl aliquot of the lysate was purified by adding 10 μl of sbeadex microbeads (from the sbeadex plant kit) and 100 μl of a mixture containing 50% isopropanol, 25% nonionic detergent, and polyethyleneimine (called Lightning mix) at pH 5. The mixture was vortexed for 10 seconds; after 1 minute, the beads were pulled aside with a magnet and the supernatant removed. The beads were then washed with 200 μl of water for 1 minute and eluted in 50 μl of elution buffer AMP for 1 minute.

[0256] Assess DNA purity and quantity on a Nanodrop. A 260 / 280 value of 1.8 to 2.0 generally indicates pure DNA / RNA free of protein.

[0257] The invention allows for up to 8 times faster DNA purification without sacrificing quality or yield, while reducing liquid waste by 50% and required consumables by 40%.

[0258] Table 10:

[0259]

[0260]

[0261] Example 11:

[0262] According to the manual, use Lysates of oak leaves were prepared using CTAB-based lysis buffer PN in the maxi plant kit.

[0263] According to Table 11, by using 100 μl of lysate and adding 10 μl Microbeads (from maxiplant kit, LGC Biosearch Technologies) and 100 μl of binding mix were used to test binding.

[0264] The sample was vortexed; after 2 minutes, the beads were pulled aside and the supernatant removed. The beads were then washed with 400 μl of water for 30 seconds and eluted in 50 μl of TRIS (pH = 9) at room temperature for 2 minutes. DNA concentration was quantified by Qubit fluorometer measurement.

[0265] From the results shown in Table 11, it can be seen that by using a lysis buffer based on a cationic detergent (CTAB), isopropanol, 20 and triethylenetetramine tetrahydrochloride (TETH) components work synergistically to bind nucleic acids and retain them on the bead surface during water washes.

[0266] Table 11:

[0267]

[0268]

[0269] Example 12:

[0270] Maize seed lysates were prepared using SDS-based lysis buffer H purchased from LGC Biosearch Technologies.

[0271] According to Table 12, by using 100 μl of lysate and adding 10 μl Microbeads (from maxiplant kit, LGC Biosearch Technologies) and 100 μl of binding mix were used to test binding.

[0272] The sample was vortexed; after 2 minutes, the beads were pulled aside and the supernatant removed. The beads were then washed with 400 μl of water for 30 seconds and eluted in 50 μl of TRIS (pH = 9) at room temperature for 2 minutes. DNA concentration was quantified by Qubit fluorometer measurement.

[0273] From the results shown in Table 12, it can be seen that by using a lysis buffer based on anionic detergent (SDS), isopropanol, The 20 and triethylenetetramine tetrahydrochloride (TETH) components act synergistically to bind nucleic acids and retain them on the bead surface during water washes. The presence of a protic alcohol is not necessary to promote binding and allow water washes.

[0274] Table 12:

[0275]

[0276] Comparative Example 13

[0277] 3 mm fish (trout) biopsy specimens were lysed with 10 μl of protease solution (20 mg / ml) in 400 μl of lysis buffer H (SDS-based lysis buffer purchased from LGC Biosearch) at 60° C. overnight. After lysis, the samples were centrifuged for 1 minute to remove unlysed sample, and 100 μl of the cleared lysate was transferred to two new tubes, 100 μl each.

[0278] To one of the test tubes (sample 1), 100 μl of binding solution (25% Tween 20, 150 mM NaAc (pH 4.5), 70% ethanol) and 10 μl of sbeadex particles purchased from LGC Biosearch were added.

[0279] To another test tube (sample 2, comparative example) were added 300 μl of binding buffer (2 M GuHCl, 10% laurylsarcosine, 30% ethanol, BisTris (pH 6.5) as disclosed in DE 102007009347A1) and 10 μl of sbeadex particles purchased from LGC Biosearch.

[0280] Vortex the tube for 30 seconds, then wait 30 seconds. Separate the beads on a magnet for approximately 50 seconds and remove the supernatant. Add 200 μl of water, vortex the tube for 30 seconds, then wait 30 seconds. Separate the beads on a magnet for approximately 10 seconds and remove the supernatant. Add 50 μl of elution buffer, vortex the tube for 30 seconds, and incubate at 60°C for 60 seconds.

[0281] Beads were separated with a magnet and DNA was analyzed by UV Vis in a nanodrop (A); and 10 μl DNA was visualized on a 0.8% agarose gel with SYBR green and 100 ng Lambda Marker (Thermo) (50 kb DNA) and NEB 1 kb DNA ladder (B).

[0282] The binding preparation disclosed in DE 102007009347 A1 does not allow purification of large amounts of DNA by water washing.

[0283] Table 13:

[0284]

[0285] *Comparative example.

Claims

1. A method for isolating and / or purifying nucleic acids from a sample, comprising: a) providing a sample containing the nucleic acid, and binding the nucleic acid to a solid phase containing carboxyl groups and / or hydroxyl groups using a binding solution, wherein the binding solution comprises: i) at least one non-ionic detergent at a concentration of at least 5% (w / v); ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and iii) an optional ligand compound comprising 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 to enable the nucleic acid to bind to the solid phase; b) separating the solid phase bound to the nucleic acid from the binding mixture; c) optionally washing the nucleic acid; as well as d) eluting the nucleic acid from the solid phase.

2. The method according to claim 1, wherein the pH for the binding step is in the range of 4 to 5. The method according to claim 1 , wherein the sample containing the nucleic acid comprises a lysis buffer.

4. The method according to one or more of claims 1 to 3, wherein the binding buffer contains i) at least one non-ionic detergent in a concentration selected from the group consisting of 5% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v) and 20% (w / v) to 25% (w / v).

5. The method according to one or more of claims 1 to 4, wherein the binding mixture comprises i) polysorbate or alcohol ethoxylate in a concentration of 20% (w / v) to 30% (w / v).

6. The method according to one or more of claims 1 to 5, 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 in a concentration selected from 50% to 75% (v / v).

8. The method according to one or more of claims 1 to 7, wherein the binding mixture comprises iii) a primary, secondary, tertiary or quaternary ammonium monoamine or polyamine or imine having at least one amino group.

9. The process according to one or more of claims 1 to 8, 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. The method according to one or more of claims 1 to 10, wherein the step of washing the nucleic acids according to step c) is carried out using pure water or an aqueous solution as washing buffer.

12. The method according to one or more of claims 1 to 11, wherein the step of washing the nucleic acids according to step c) is carried out with pure water.

13. The method according to one or more of claims 1 to 12, wherein the step of eluting the nucleic acids 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; b) a binding solution comprising: i) at least one non-ionic detergent at a concentration of at least 5% (w / v); ii) Optional linear or branched C 1-4 Aqueous solutions of alcohol or acetone; and iii) an optional ligand compound comprising at least one protonatable group, wherein the binding buffer has a pH selected from the group consisting of ≤6, ≤5.5, ≤4, and ≥3; c) a solid phase comprising carboxyl and / or hydroxyl groups, said solid phase being capable of selectively binding to said nucleic acid in the presence of said ligand compound; d) optionally one or more washing solutions; and e) optionally one or more elution solutions.

15. A preparation for use in the method according to one or more of claims 1 to 13, comprising: i) at least one non-ionic detergent at a concentration of 5% (w / v) to 50% (w / v), 10% (w / v) to 30% (w / v), and 20% (w / v) to 25% (w / v); ii) 50% to 75% ethanol or isopropyl alcohol; 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 their corresponding salts; and iv) acidifying and / or buffering compounds for adjusting the pH of the formulation to between 3 and 6.

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

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