Methods of isolating and / or purifying nucleic acids

Abstract

The present disclosure relates to a nucleic acid purification method comprising: (a) combining a sample comprising at least one nucleic acid with a binding buffer comprising at least one silica silicon-based magnetic particle and having a pH of from 5 to about 10 to form a solution; (b) incubating the sample with a binding buffer for a time sufficient to reversibly bind the at least one nucleic acid to the magnetic particles to form at least one modified magnetic particle; (c) separating the at least one modified magnetic particle from the solution; (d) washing the at least one modified magnetic particle with at least one wash buffer; and (e) combining the at least one modified magnetic particle with a low conductivity elution buffer to elute the purified nucleic acid from the magnetic particle. Also disclosed herein are kits comprising these buffers and magnetic particles.

Description

Technical Field

[0001] The present invention relates to general methods and kits for nucleic acid purification, and more particularly to kits and methods based on silica-based magnetic particles for purifying samples containing RNA. Background Art

[0002] Nucleic acid purification and isolation are important steps in various biochemical and diagnostic procedures. In vitro transcription (IVT) can provide a method to transcribe nucleotides with desired sequences or modifications in vitro. The mRNA obtained by IVT can be further used in RNA-based therapeutics, such as vaccine formulations. However, before downstream applications, the post-IVT sample needs to be purified to remove one or more contaminants, such as salts, proteins, enzymes, oligonucleotides, etc. The presence of such contaminants can hinder or prevent many downstream processes. Therefore, it is very important to effectively separate nucleic acids from the post-IVT mixture to ensure the desired end-use functionality.

[0003] Nucleic acid purification methods are described in the art. Conventional precipitation procedures (e.g., phenol / chloroform extraction) yield highly purified RNA, but are time-consuming and complex. Solid-phase based methods (e.g., methods utilizing magnetic beads, spin columns, and / or filtration systems) have been proposed as an alternative solution. However, the above methods also have some disadvantages, such as slow speed, high complexity, and low overall yield.

[0004] Methods for purifying nucleic acids safely for use as drugs are more limited. Current RNA vaccine production protocols use expensive techniques such as chromatography or tangential flow filtration for purification steps. Magnetic bead-based RNA purification processes are also used in the field of RNA production, but are mainly used for analytical purposes because the process requires the use of toxic and expensive substances such as the chaotropic agent guanidine thiocyanate and solvents such as isopropanol, which limits its accessibility and utilization in RNA therapy.

[0005] In order to provide more RNA-based drugs, a purification method that is efficient, cheap and compliant with drug safety regulations is needed. The present invention aims to provide a solution to this problem. Summary of the invention

[0006] The present invention and its embodiments are intended to provide solutions to one or more of the above-mentioned disadvantages and describe a new bead-based method for purification of nucleic acid sequences using commonly available, non-hazardous and economical materials / techniques.

[0007] In various embodiments, the present disclosure relates to a method for purifying nucleic acid. In one embodiment, the method is the method disclosed in claim 1.

[0008] The method comprises: (a) mixing a sample comprising at least one target nucleic acid with silica-based magnetic particles in a binding buffer having a pH of about 5 to about 10 to form a solution, (b) incubating the solution for a time sufficient to reversibly bind the at least one nucleic acid to the magnetic particles to form modified magnetic particles, (c) separating the modified magnetic particles from the combined solution by applying a magnetic field, (d) washing the at least one modified magnetic particle with at least one wash buffer; and (e) combining the modified magnetic particles with an elution buffer to elute the target nucleic acid from the magnetic particles, wherein the pH of the elution buffer is about 5 to 10, and wherein the conductivity of the elution buffer is 0.001 mS to 40 mS / cm, or wherein the total salt concentration is 0 mM to 50 mM.

[0009] Further embodiments are described in the dependent claims.

[0010] According to different embodiments, the binding buffer comprises at least one salt having a concentration of about 0.1 M to about 5 M, preferably a salt safe for human use, more preferably table salt, and at least one alcohol having a concentration of about 10% to 50% volume / volume (v / v), and / or polyethylene glycol (PEG) having a concentration of 10% to 40% (v / v).

[0011] In other embodiments, the wash buffer may comprise at least one alcohol at about 60% to 80% v / v and optionally at least one buffering compound, such as Tris-HCl or sodium citrate.

[0012] The elution buffer is preferably selected from water and a solution comprising at least one buffer compound, such as Tris-HCl or sodium citrate and / or at least one ion chelator, such as EDTA, etc. The silica-based magnetic particles may be coated with one or more ligands interacting with the nucleic acid and combinations thereof.

[0013] Also disclosed herein is a nucleic acid purification kit comprising the buffers and magnetic particles as described in claim 19. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The following shows the workflow of the nucleic acid purification method according to the embodiment of the present invention.

[0015] Figure 2 and Figure 3 Two exemplary processes of the nucleic acid purification method according to the embodiment of the present invention are shown.

[0016] 4 and 5 show a set of graphs showing successful purification of high quality RNA using binding buffers of varying NaCl concentrations and pH according to the disclosed method. DETAILED DESCRIPTION

[0017] definition

[0018] Unless otherwise defined, all terms used in the present disclosure, including technical and scientific terms, have the meanings commonly understood by ordinary technicians in the field to which the present disclosure belongs. By way of further guidance, term definitions are included to better understand the teachings of the present disclosure.

[0019] As used herein, the following terms have the following meanings:

[0020] Unless the context clearly indicates otherwise, "a", "an" and "the" as used herein refer to both singular and plural referents. For example, "a compartment" refers to one or more than one compartment.

[0021] As used herein, "about" refers to a measurable value such as a parameter, quantity, duration, etc., and is intended to include variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less from the specific value, as long as it is suitable to implement these variations in the disclosure. However, it should be understood that the value referred to by the modifier "about" itself is also specifically disclosed.

[0022] As used herein, "comprising" is synonymous with "including" and is an inclusive or open term that specifies the presence of subsequent content (e.g., components) but does not preclude the presence of other additional, unlisted components, features, elements, elements, steps known or disclosed in the art.

[0023] In addition, the terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order unless otherwise indicated. It should be understood that the terms so used are interchangeable under appropriate conditions, and that the embodiments described herein are capable of operating in a sequence other than that described or illustrated herein.

[0024] The recitation of numerical values ​​by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0025] Unless otherwise defined, the expressions "weight %", "weight percent", "% wt" or "wt %" here and throughout the specification refer to the relative weight of the corresponding component based on the total weight of the formulation.

[0026] Although the term "one or more" or "at least one", such as one or more or at least one of a group of elements, is clear in itself, by further example, the term includes reference to any one of the elements, or to any two or more of the elements, such as any ≥3, ≥4, ≥5, ≥6 or ≥7, etc., of the elements, as well as to all of the elements.

[0027] As used herein, the term "magnetic particle" and variations thereof are intended to mean a particle having a magnetic (eg, paramagnetic or superparamagnetic) core coated with at least one material having a surface capable of reversibly binding to nucleic acids.

[0028] The terms "magnetic particles" and "magnetic beads" are used interchangeably.

[0029] The term "biological sample" refers to a sample from a biological source, for example, bacteria and other microorganisms, bacteria, from plants, humans or non-human animals, preferably warm-blooded animals, even more preferably mammals, for example, non-human primates, rodents, canines, felines, equines, ovines, swine, etc. The term "non-human animals" includes all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals, such as chickens, amphibians, reptiles, etc. Preferably, the biological sample is from a human. The biological sample can be a biological fluid or a non-fluid biological sample.

[0030] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. By way of further guidance, definitions of terms are included in this specification to better understand the teachings of this disclosure. Terms or definitions used herein are only intended to aid understanding of this disclosure.

[0031] References throughout this specification to "a certain embodiment" and "an embodiment" indicate that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the phrases "in a certain embodiment" and "in an embodiment" that appear throughout this specification do not necessarily all refer to the same embodiment, but may do so. In addition, as will be apparent to those skilled in the art from the present invention, in one or more embodiments, particular features, structures, or characteristics may be combined in any suitable manner. In addition, although some embodiments described herein include some features included in other embodiments without including other features, combinations of features from different embodiments should also fall within the scope of the present disclosure and form different embodiments, as understood by those skilled in the art. For example, in the following claims, any claimed embodiment may be used in any combination.

[0032] Detailed Description

[0033] The present invention aims to provide methods and kits for purifying nucleic acids using magnetic particles, wherein once eluted, the nucleic acids are already in a buffer suitable for further processing and formulation, without any further buffer exchange. These methods and kits purify the nucleic acids (economically) efficiently. The bead-based purification methods disclosed herein produce RNA-based drugs that meet the requirements of the authorities by utilizing common, harmless and economical substances / techniques. In order to carry out these processes, it is preferred to use common salts such as sodium chloride, buffers such as Tris-HCl or citrate, and limited amounts of solvents. The methods described herein allow purification of nucleic acids with high yields (in the range of 80% to 100%) and sufficient purity. The integrity of the RNA is retained, while the use of harmful preparations is reduced, and the technology is economical.

[0034] In a first aspect, the present invention relates to a method for purifying nucleic acid. In one embodiment, the method comprises:

[0035] a) combining a sample comprising at least one target nucleic acid in a binding buffer at a pH of 5 to 10 with silica-based magnetic particles to form a solution;

[0036] b) incubating the solution for a time sufficient to reversibly bind the at least one nucleic acid to the magnetic particles to form modified magnetic particles;

[0037] c) separating the modified magnetic particles from the combined solution by applying a magnetic field;

[0038] wherein the binding buffer comprises at least one salt having a concentration of 0.1M to 5M, at least one first alcohol having a concentration of 10% to 50% v / v and / or PEG having a concentration of 10% to 40% v / v,

[0039] d) washing the at least one modified magnetic particle with at least one wash buffer comprising at least one second alcohol at a concentration of 60% to 100%; and

[0040] e) combining the modified magnetic particles with an elution buffer to elute the target nucleic acid from the magnetic particles, wherein the pH of the elution buffer is about 5 to 10 and the conductivity of the elution buffer is 0.001 mS to 40 mS / cm, or the total salt concentration is 0 mM to 50 mM.

[0041] It will be appreciated that separation of the magnetic particles from the solution also requires physical separation, such as removal of the supernatant from the modified magnetic particles.

[0042] In embodiments, washing of the modified particles may be repeated multiple times, such as 2 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, and 10 times.

[0043] It will be clear to those skilled in the art that washing requires adding and removing wash buffer to the modified beads. In each successive wash step, before the next wash and / or before any other subsequent step (e.g., elution step), the modified particles are separated from the wash buffer by magnetic separation.

[0044] It will also be clear to those skilled in the art that in an embodiment, the elution step is repeated multiple times to increase the yield, for example, the elution is repeated 2, 4, 5, 6, 7, 8, 9 and 10 times.

[0045] Although the step-by-step purification scheme is similar to the prior art, the method herein uses a new buffer combination, which avoids the use of toxic or unnecessary compounds (e.g., guanidine salts) commonly used in the prior art, improves yield, reduces costs, and shortens the time to complete RNA purification. This effect is mainly achieved by selecting a specific type of magnetic particles (i.e., silica-based magnetic particles). The silica-based magnetic particles allow the use of generally less harsh binding conditions, especially elution conditions, while still ensuring high yield and high purity of the nucleic acid to be purified. The resulting purified sample can be immediately used for further downstream pharmaceutical preparations or for the production of pharmaceutical preparations without further downstream purification to remove the components used in the binding and / or elution buffers. This is very different from the known prior art methods (e.g., using cellulose or cellulose derivatives).

[0046] The method is particularly advantageous for the efficient purification of nucleic acids, separating them from harmful contaminants (e.g., salts, proteins, enzymes, oligonucleotides, carbohydrates, DNA templates, and nucleotide triphosphates (NTPs)) in the sample. The method purifies the nucleic acid, wherein the nucleic acid is RNA, more specifically in vitro transcribed RNA, with high yield (80% to 100%) and sufficient purity, and retains the integrity of the purified RNA while reducing the use of dangerous and expensive reagents, such as chaotropic agents commonly used in practice, such as guanidine thiocyanate. Overall, the technology is safe and economical.

[0047] In a specific embodiment, the method may comprise adding a further sample comprising at least one target nucleic acid in a binding buffer and optionally unmodified silica-based magnetic particles to the modified magnetic particles of the preceding step c to form a solution, and repeating steps b and c. Thus, the methods disclosed herein comprise repetitions of some consecutive steps.

[0048] In an embodiment, the steps of the method of the present invention comprising sample-magnetic particle binding (steps a and b) and the continuous magnetic separation step (hereinafter referred to as the "binding step") can be repeated multiple times in succession, and then the following steps comprising washing and elution are performed. Therefore, in an embodiment, before performing step d, steps a to c of the present method can be repeated once or multiple times, preferably repeated 1 to 20 times, more preferably repeated 1 to 10 times. In an embodiment, the binding step is repeated continuously up to 20 times, up to 15 times, up to 10 times, up to 9 times, up to 8 times, up to 7 times, up to 6 times, up to 5 times, up to 4 times, up to 3 times, up to 2 times, preferably repeated 5 to 15 times. The inventors have found that repetition can reduce the amount of alcohol such as ethanol required per unit of purified nucleic acid.

[0049] In embodiments, the cumulative repeated binding steps can reduce the total alcohol consumption per unit nucleic acid by 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% compared to a method using a single binding step. In embodiments, the alcohol can be ethanol.

[0050] According to embodiments, when continuous binding steps are applied, the reduction in the amount of alcohol used can be due to the reduction in the amount of alcohol (e.g., ethanol) used in the washing steps of the disclosed methods. In embodiments, cumulative repeated binding steps can reduce the total alcohol consumption per unit nucleic acid by up to 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% compared to methods using a single binding step. In embodiments, the alcohol can be ethanol.

[0051] In an embodiment, these continuous binding steps can be performed by continuously adding magnetic particles and nucleic acids in each cycle (hereinafter referred to as the first option) or by placing all magnetic particles in a tube in one step and continuously adding nucleic acids in each cycle (hereinafter referred to as the second option). When the second option is selected, the magnetic particles added in one step should have a sufficient concentration to have sufficient binding capacity for the total amount of nucleic acids added in subsequent continuous cycles. It is obvious to those skilled in the art that in the second option, the ratio of the concentration of magnetic particles to nucleic acids in the first cycle of the continuous binding step is higher than that in the first option. In an embodiment, for purifying the same amount of nucleic acids, the concentration of magnetic particles added in the first cycle of the continuous binding step in the second option is 20 times higher than that in the first option. For example, the second option is 15 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times higher than the first option.

[0052] In a specific embodiment, the method comprises an additional step after step c and before step d, wherein the additional step is

[0053] f) adding at least one target nucleic acid in a binding buffer to the modified magnetic particles;

[0054] g) separating the modified magnetic particles from the combined solution by applying a magnetic field;

[0055] Wherein, before carrying out steps d and e, steps f and g are sequentially repeated once or multiple times, preferably repeated 10 times.

[0056] More specifically, in this embodiment, the method may include:

[0057] a) combining a sample comprising at least one target nucleic acid with silica-based magnetic particles in a binding buffer at a pH of 5 to 10 to form a solution;

[0058] b) incubating the solution for a time sufficient to reversibly bind the at least one nucleic acid to the magnetic particles to form modified magnetic particles;

[0059] c) separating the modified magnetic particles from the combined solution by applying a magnetic field;

[0060] f) adding at least one target nucleic acid in a binding buffer to the modified magnetic particles;

[0061] g) separating the modified magnetic particles from the combined solution by applying a magnetic field;

[0062] wherein the binding buffer comprises at least one salt having a concentration of 0.1M to 5M, at least one first alcohol having a concentration of 10% to 50% v / v and / or PEG having a concentration of 10% to 40% v / v,

[0063] wherein steps i and j are repeated consecutively up to 20 times, preferably up to 10 times,

[0064] d) washing the at least one modified magnetic particle with at least one wash buffer comprising at least one second alcohol in a concentration of 50% to 100%; and

[0065] combining the modified magnetic particles with an elution buffer to elute the target nucleic acid from the magnetic particles,

[0066] wherein the pH of the elution buffer is about 5 to 10, and wherein the conductivity of the elution buffer is 0.001 mS to 40 mS / cm, or wherein the total salt concentration is 0 mM to 50 mM.

[0067] In embodiments of the methods taught herein, a sample comprising at least one nucleic acid can be combined with a binding buffer.

[0068] In embodiments, the pH of the binding buffer may be from 5 to 10, e.g., from 5 to 9, from 5.5 to 8.5, from 6 to 8, or from 6.4 to 7.5, and ranges and subranges therebetween. In embodiments, the binding buffer comprises at least one first alcohol and / or PEG, at least one salt, and at least one optional chelating agent, e.g., EDTA.

[0069] The concentration of the at least one first alcohol in the binding buffer is 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 20% v / v, including all ranges and subranges therebetween.

[0070] According to various embodiments, at least one alcohol may be selected from isopropanol, ethanol, methanol, butanol and combinations thereof. In some embodiments, at least one alcohol may be ethanol, which is more suitable for the purpose of producing drugs than isopropanol commonly used in the prior art.

[0071] The concentration of PEG in the binding buffer can be 10% to 40%, 20% to 40%, 20% to 35%, 20% to 30%, or 25% to 35%, including all ranges and subranges therebetween. In one embodiment, 30% PEG is used.

[0072] In one embodiment, the PEG used in the binding buffer is selected from PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 6000, PEG 8000, PEG 10.000, PEG 20.000. In one embodiment, the PEG used is PEG 8000.

[0073] At least one salt may be present in the binding buffer at a concentration of 0.1 M to 5 M, e.g., 0.1 M to 4 M, 0.1 M to 3 M, 0.1 M to 2 M, 0.1 M to 1 M, 0.5 M to 1 M, 0.5 M to 2 M, 1 M to 2 M, 2 M to 3 M, and 3 M to 5 M, including all ranges and subranges therebetween.

[0074] In embodiments, at least one salt may be present in the binding buffer at a concentration of 0.1 M to 0.5 M, 0.5 M to 1.5 M, 0.5 M to 5 M, 1 M to 5 M, 2 M to 5 M, 3 M to 5 M, 4 M to 5 M, 3 M to 4 M, 1 M to 3 M, 1 M to 4 M, 1 M to 1.5 M, 1 M to 1.6 M, 1 M to 1.7 M, 1 M to 1.8 M, including all ranges and subranges therebetween.

[0075] According to various embodiments, the at least one salt may be sodium chloride (NaCl).

[0076] In an embodiment, the binding buffer does not contain toxic chaotropic agents, such as guanidine salts (guanidinium thiocyanate or guanidine thiocyanate), iodides, perchlorates and trichloroacetates, preferably guanidine salts. Most methods in the prior art (including magnetic bead purification) use guanidine salts (such as guanidine thiocyanate or guanidine hydrochloride) as commonly used substances because these chemicals have extremely strong chaotropic properties. In an embodiment of the method taught herein, sodium chloride (NaCl) is used instead of highly toxic and expensive guanidine thiocyanate, which brings advantages in many aspects to the present invention, such as reducing costs, making the working environment safer, complying with regulations for safe drug production, and accelerating RNA purification due to reducing the number of washing steps to remove toxic guanidine thiocyanate from the sample.

[0077] The term "magnetic particle" and variants thereof as used herein are intended to refer to particles having a magnetic (e.g., paramagnetic or superparamagnetic) core coated with at least one material having a surface capable of reversibly binding to nucleic acids. Suitable magnetic particles may include, for example, carboxyl-coated paramagnetic particles, silica-based paramagnetic particles, etc. In embodiments described herein, the magnetic particles are silica-based magnetic particles, wherein in some embodiments, the silica-based particles may include a paramagnetic core coated with silica, thereby providing a hydrated silica adsorption surface (e.g., a surface comprising silanol groups) for nucleic acid binding. In other or further embodiments, the magnetic particles may be coated with a ligand that interacts with nucleic acids, or may be surface modified to produce a functionalized surface, such as a weakly or strongly positively charged, a weakly or strongly negatively charged, or a hydrophobic surface, to name a few.

[0078] The concentration of the magnetic particles in the binding buffer can be, for example, about 0.1 μg / μl to about 60 μg / μl, for example, about 0.5 μg / μl to about 55 μg / μl, about 1 μg / μl to about 50 μg / μl, 0.1 μg / μl to 20 μg / μl, 1 μg / μl to 15 μg / μl, 5 μg / μl to 10 μg / μl, about 2 μg / μl to about 45 μg / μl. g / μl, about 3 μg / μl to about 40 μg / μl, about 4 μg / μl to about 35 μg / μl, about 5 μg / μl to about 30 μg / μl, about 6 μg / μl to about 25 μg / μl, about 7 μg / μl to about 20 μg / μl, about 8 μg / μl to about 15 μg / μl, or about 9 μg / μl to about 10 μg / μl, including all ranges and subranges therebetween.

[0079] In a specific embodiment, for example, in the case where the binding step is preferably repeated, additional magnetic particles may be added to the binding buffer. Obviously, further addition of magnetic beads will increase the final concentration of magnetic beads in the binding buffer. In an embodiment, as disclosed above, the additional magnetic particles added to the binding buffer may result in a concentration of magnetic beads in the binding buffer of 20 times, for example 15 times, 10 times, 5 times, 2 times, of 60 μg / μl. For example, the final concentration of magnetic particles in the binding buffer is 0.1 μg / μl to 1200 μg / μl, for example 1 μg / μl to 1100 μg / μl, 10 μg / μl to 1000 μg / μl, 20 μg / μl to 800 μg / μl, 30 μg / μl to 700 μg / μl, 40 μg / μl to 600 μg / μl, 40 μg / μl to 500 μg / μl, 50 μg / μl to 600 μg / μl, 50 μg / μl to 700 μg / μl, 50 μg / μl to 800 μg / μl, 50 μg / μl to 900 μg / μl, 50 μg / μl to 900 μg / μl, 50 μg / μl to 10 ... g / μl to 400μg / μl, 60μg / μl to 300μg / μl, 70μg / μl to 200μg / μl, 80μg / μl to 150μg / μl, 600μg / μl to 1200μg / μl, 700μg / μl to 1200μg / μl, 800μg / μl to 1200μg / μl, 900μg / μl to 1200μg / μl, 1000μg / μl to 1200μg / μl or 0.1μg / μl to 1200μg / μl, 0.1μg / μl to 1100μg / μl, 0.1μg / μl to 1000μg / μl, 0.1μl g / μl to 900μg / μl, 0.1μg / μl to 800μg / μl, 0.1μg / μl to 700μg / μl, 0.1μg / μl to 600μg / μl, 0.1μg / μg / μl to 500 μg / μl, 0.1 μg / μl to 400 μg / μl, 0.1 μg / μl to 300 μg / μl, 0.1 μg / μl to 200 μg / μl, 0.1 μg / μl to 100 μg / μl, 1 μg / μl to 100 μg / μl, 10 μg / μl to 100 μg / μl, or 50 μg / μl to 100 μg / μl, including all ranges and subranges therebetween.

[0080] In embodiments of the methods taught herein, the volume ratio between the sample and the binding buffer can be, for example, 1:1 to 1:3, such as 1:1 to 1:1.5, or 1:1.5 to about 1:2.5, including all ranges and sub-ranges therebetween. The incubation time of the mixed solution comprising a sample containing at least one target nucleic acid, a binding buffer and silica-based magnetic particles can be 0.1 minute to 30 minutes, 0.1 minute to 25 minutes, 0.1 minute to 20 minutes, 0.1 minute to 10 minutes, or 0.1 minute to 5 minutes, 0.1 minute to 2 minutes, including all ranges and sub-ranges therebetween.

[0081] In a specific embodiment, for example, in the case of preferably repeating the binding step, the volume ratio between the sample further added in the repeating step and the binding buffer can be, for example, 1: 1 to 1: 3, for example 1: 1 to 1: 1.5, or 1: 1.5 to about 1: 2.5, including all ranges and sub-ranges therebetween. Obviously, when preferably repeating the binding step, most of the magnetic beads and most of the samples added in the first few rounds of binding will remain in the tubes of further samples to which the binding buffer has been added. It is expected that most of the samples added in the first few rounds of binding steps will be attached to the magnetic particles. For the technician, it is obvious that the ratio of the overall sample in the reaction tube (including the samples added in all the rounds of binding steps and the samples further added at the end) to the binding buffer will be higher than the above ratio. The volume ratio between the total sample and the binding buffer can be, for example, 1:3 to 20:1, such as 1:3 to 1:2, 1:2 to 1:1, 1:3 to 20:1, 1:3 to 19:1, 1:3 to 18:1, 1:3 to 17:1, 1:3 to 16:1, 1:3 to 15:1, 1:3 to 14:1, 1:3 to 13:1, 1:3 to 12:1, 1:3 to 11:1, 1:3 to 10:1, 1:3 to 9:1, 1:3 to 8:1, 1:3 to 7:1, 1:3 to 6:1, 1:3 to 4:1, 1:3 to 3:1, 1:3 to 2:1, including all ranges and subranges therebetween.

[0082] It is believed that the salts and alcohols introduced by the binding buffer can enhance the ability of nucleic acids (e.g., RNA) to reversibly (e.g., non-covalently) bind to the surface of magnetic particles (e.g., silica surface). The magnetic particles modified in this way (e.g., containing reversibly bound nucleic acids) can then be separated from unbound contaminants (e.g., salts, proteins, enzymes, oligonucleotides, DNA templates, and nucleoside triphosphates (NTPs)). For example, a magnet can be placed near the modified magnetic particles to attract the particles together, such as to form aggregates or particles. In certain embodiments, a container (e.g., a tube) containing a combined solution containing modified magnetic particles can be placed on a magnetic stand, which can aggregate the particles and fix the particles to a certain extent while removing the remaining solution.

[0083] After the nucleic acid is combined with the magnetic particles and the modified magnetic particles are separated using a magnet, the particles can then be combined, rinsed or washed with one or more wash buffers. The wash buffer can contain, for example, at least one alcohol, optionally at least one buffer compound. The modified magnetic particles can be rinsed with wash buffer one or more times, for example once, twice, three times or more. Any additional washing can use the same or different compositions, concentrations and / or volumes.

[0084] In embodiments of the methods taught herein, the wash buffer comprises at least one second alcohol at a concentration of, for example, 50% to 100% volume / volume (v / v), 55% to 95%, 60% to 85%, or 60% to 80% (v / v), including all ranges and sub-ranges therebetween. In embodiments, at least one second alcohol may be selected from isopropanol, methanol, ethanol, butanol, and combinations thereof. At least one second alcohol in the wash buffer may be the same or different from a first alcohol in the binding buffer. In some embodiments, at least one second alcohol may be ethanol. In non-limiting embodiments, the wash buffer may optionally have at least one salt. The concentration of the optional at least one salt (if present) in the binding buffer may be 0.1M to 5M, for example 0.1M to 4M, 0.1M to 3M, 0.1M to 2M, 0.1M to 1M, and 1M to 2M, including all ranges and sub-ranges therebetween.

[0085] The concentration of the optional at least one salt in the binding buffer, if present, is from 0.5M to 5M, e.g., from 1M to 5M, 2M to 5M, 3M to 5M, 4M to 5M, 2M to 4M, 3M to 4M, 2M to 3M and from 1M to 4M, including all ranges and subranges therebetween.

[0086] According to various embodiments, at least one salt in the wash buffer may be sodium chloride (NaCl). In non-limiting embodiments, the wash buffer may optionally have at least one buffer and / or salt compound (e.g., Tris, citrate, etc.) at a concentration of 1 mM to about 10 mM. In embodiments, the modified particles may be washed one or more times with at least one of the wash buffers. For example, the modified magnetic particles may be washed once, twice, or more times with a wash buffer, and the modified magnetic particles may be separated using a magnet in the intervals between washes.

[0087] After the washing steps, after addition and removal of the washing buffer, modified magnetic particles with nucleic acids reversibly bound to the surface can be provided, which are free or substantially free of contaminants such as salts, proteins, enzymes, etc. According to various embodiments, the modified magnetic particles thus produced can then be combined with one or more elution buffers to release the bound nucleic acids and separate them from the magnetic particles.

[0088] According to various embodiments, the elution buffer is a low conductivity solution, wherein the conductivity of the buffer is 0.001 mS / cm to 40 mS / cm, more preferably 0.01 mS / cm to 40 mS / cm, 0.1 mS to 40 mS / cm, 0.5 mS / cm to 40 mS / cm, more preferably 0.5 mS / cm to 30 mS / cm, 0.5 mS / cm to 20 mS / cm, 0.5 mS / cm to 10 mS / cm, including all ranges and sub-ranges therebetween.

[0089] In another or further embodiment, the elution buffer comprises a salt concentration of 0.01 mM to 50 mM, more preferably 0.1 mM to 40 mM, more preferably 0.1 mM to 30 mM, more preferably 0.1 mM to 20 mM.

[0090] Possible salts include sodium citrate, sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and combinations thereof.

[0091] The pH of the elution buffer can be, for example, from 5 to about 10, such as 5.5 to about 9, 6 to 8, or 6.4 to about 7.5, including all ranges and subranges therebetween.

[0092] For example, in some cases, the elution buffer comprises water; in other cases, the elution buffer comprises water and EDTA, or only Tris, or Tris and EDTA, or sodium citrate, or phosphate buffer, or phosphate buffered saline (PBS). If used as elution buffer, the concentration of sodium citrate can be 0.5mM to 10mM, such as 0.6mM to 5mM, 1mM to 2mM, including all ranges and subranges therebetween. If used as elution buffer, the pH of sodium citrate can be pH5.4 to 7.5, pH 6 to pH 7, pH 6 to pH 6.5, including all ranges and subranges therebetween. According to non-limiting embodiments, the elution buffer can comprise water or 10mM Tris-HCl, 1mM EDTA, pH 7.4; or 10mM Tris-HCl, pH 7.4; or 1mM sodium citrate, pH 6.4.

[0093] In an embodiment, the elution buffer does not contain toxic chaotropic agents such as guanidinium salts (guanidinium thiocyanate or guanidine thiocyanate), iodide, perchlorate and trichloroacetate, preferably guanidinium salts.

[0094] In embodiments, the at least one modified particle is incubated with the elution buffer for 30 seconds to 30 minutes, 1 minute to 20 minutes, 1 minute to 10 minutes, including all ranges and subranges therebetween.

[0095] The magnetic particles can then be removed from the solution (no longer bound to the nucleic acid), for example, by separation with a magnet, thereby obtaining the purified nucleic acid in the solution as a final product. The modified magnetic beads can be incubated with the elution buffer for several rounds (e.g., once, twice or more) to optimally elute the purified nucleic acid. For example, the method disclosed herein can be used to provide a purified RNA product. In certain embodiments, the method disclosed herein can provide a relative RNA yield of 80% to 100%, and provide sufficient purity. In the embodiments of the method taught herein, the purified RNA integrity is retained while reducing the use of harmful agents and making the technology economical by reducing costs.

[0096] In embodiments of the methods taught herein, the target nucleic acid may be a DNA molecule, an RNA molecule, or a DNA / RNA hybrid molecule. The at least one nucleic acid may include, for example, genomic DNA, chromosomal DNA (cDNA), plasmid DNA (pDNA), total RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), small RNA, and / or RNA / DNA hybrids. In various embodiments, the sample may be a PCR product comprising DNA. In some embodiments, the sample may be an in vitro transcribed (IVT) mixture comprising RNA, wherein the IVT RNA may be 5' capped or uncapped RNA. In various embodiments, the sample comprising at least one nucleic acid may be extracted from a biological sample, wherein the biological sample may be of bacterial, viral, plant, or animal origin. According to other embodiments, the at least one nucleic acid to be purified may be RNA, such as total RNA.

[0097] Therefore, the present disclosure also relates to an in vitro transcription method for RNA, wherein the obtained RNA is subjected to at least one silica magnetic bead purification step according to any embodiment described herein.

[0098] In embodiments of the methods taught herein, the methods may be semi-automated or fully automated.

[0099] In an embodiment of the method taught herein, the method can be used to produce a kit for nucleic acid purification, wherein the kit comprises a binding buffer, a washing buffer and an elution buffer. In a preferred embodiment, a kit for nucleic acid purification is disclosed, comprising:

[0100] a) a binding buffer having a pH of 5 to 10; at least one salt, being NaCl, having a concentration of 0.1 M to 5 M; ethanol having a concentration of 10% to 50% v / v; and / or PEG having a concentration of 10% to 40% v / v.

[0101] The proportion of ethanol herein is preferably 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, more preferably 15% to 20% v / v, including all ranges and sub-ranges therebetween.

[0102] The concentration of PEG in the binding buffer is preferably 10% to 40%, 20% to 40%, 20% to 35%, 20% to 30%, or 25% to 35%, including all ranges and subranges therebetween.

[0103] b) a wash buffer comprising at least 60% by volume of ethanol and optionally at least one buffer and / or salt compound.

[0104] c) an elution buffer selected from water, a solution comprising at least one buffer compound, the conductivity of which is between 0.001 mS and 40 mS / cm, or wherein the total salt concentration is between 0 mM and 50 mM.

[0105] In one embodiment, the kit further comprises a magnetic particle solution. In an embodiment, the buffers in the kit, in particular the binding buffer and the elution buffer, do not comprise toxic chaotropic agents, such as guanidine salts (guanidinium thiocyanate or guanidine thiocyanate), iodides, perchlorates and trichloroacetates, preferably guanidine salts.

[0106] It will be clear to those skilled in the art that the embodiments described above in the context of methodology are also applicable to the kits described herein and are incorporated herein.

[0107] Description of the embodiments and drawings

[0108] Figure 1 The flowchart of the nucleic acid purification method according to the embodiment of the present invention is shown in the order of steps (binding, washing and elution).

[0109] Figure 2 The schematic process of the nucleic acid purification method according to the embodiment of the present invention is shown. The process comprises:

[0110] - a binding step, in which the coated silica magnetic particles, the IVT sample with nucleic acid and a binding buffer are mixed together. The resulting mixture is incubated to allow the nucleic acid to reversibly bind to the magnetic particles;

[0111] - a magnetic separation step, in which a magnetic field is applied to separate the magnetic particles from the nucleic acids;

[0112] - a washing step, wherein a washing buffer comprising an alcohol (eg ethanol) is added and the magnetic field is applied again to separate the magnetic particles from the washing buffer, which is then removed. The washing step can be applied once, twice or more.

[0113] - Finally, the process involves an elution step, in which an elution buffer is added to release the nucleic acids from the magnetic particles, and a magnetic field is applied to separate the magnetic particles from the eluate containing the nucleic acids. The elution step can be applied once, twice or multiple times.

[0114] Figure 3 The process of the nucleic acid purification method according to the embodiment of the present invention is shown. In this embodiment, the binding step and the magnetic separation step are repeated up to 10 times before proceeding to the next step. The washing step and the elution step can be repeated once, twice or multiple times.

[0115] FIG4 shows a set of bar graphs showing successful high quality RNA purification using binding buffers of varying NaCl concentrations and pH according to the disclosed methods. 4A) RNA / protein ratio is not affected by the range of binding buffer salts. 4B) RNA / protein ratio is not affected by the pH range of the binding buffer.

[0116] FIG5 shows a set of bar graphs showing successful high quality RNA purification using binding buffers of varying NaCl concentrations and pH according to the disclosed methods. 4A) rDNA content is not affected by the salt range of the binding buffer. 4B) rDNA content is not affected by the pH range of the binding buffer. rDNA (residual DNA), AMP+ (ampicillin resistance gene), GOI-G4 (target gene).

[0117] Example 1

[0118] Non-limiting examples of possible buffers for use in conjunction with the methods described herein include:

[0119] ● Binding Buffer:

[0120] 1.13mM Tris-HCl, 1.61M NaCl, 1.3mM EDTA, pH 7.4, 17% EtOH (or 13%

[0121] EtOH)

[0122] 2.4M NaCl, 10mM Tris-HCl, 1mM EDTA, 30% PEG 8000, pH 8.0

[0123] · Wash Buffer:

[0124] 1. Ethanol 60% to 80%

[0125] 2.10mM Tris-HCl, 1mM EDTA, 70% EtOH

[0126] · Elution buffer : Choose from the following solutions

[0127] 1. Water or

[0128] 2.10mM Tris-HCl, 1mM EDTA, pH 7.4 or

[0129] 3.5 mM Tris-HCl, pH 8.0 or

[0130] 4.10 mM Tris-HCl, pH 7.4 or

[0131] 5.1 mM sodium citrate, pH 6.4

[0132] Example 2

[0133] Bead preparation

[0134] 3.5 ml of RNA solution (2 mg RNA / ml) was mixed with an appropriate amount of silica magnetic beads with a binding capacity of approximately 430 μg RNA / mg beads.

[0135] In the pretreatment step, the beads were washed with RNAse-free water. Briefly, RNAse-free water was added to the beads, the solution was vortexed, and a magnetic field was applied for 120 seconds. The supernatant was then removed.

[0136] Subsequently, the beads were washed three times with a buffer containing 40 mM Tris-HCl, 4.8 M NaCl, 4 mM EDTA at pH 7.4. The solution was vortexed and a magnetic field was applied for 120 seconds before removing the supernatant.

[0137] RNA Binding Step

[0138] Mix the RNA solution from IVT with alcohol-free binding buffer and silica magnetic beads. After homogenizing the solution, add 100% EtOH so that the final volume is 17% EtOH. Mix the solution well for binding. Incubate for at least 2 minutes. Binding is performed at room temperature.

[0139] In the subsequent step, the beads were placed in a magnetic field and the supernatant was removed. In the next step, the RNA-bound beads were washed twice with cold 80% EtOH.

[0140] In an embodiment, the RNA-bound beads are washed with cold EtOH at a concentration of 50% to 100% EtOH, e.g., 55% to 100% EtOH, 60% to 100% EtOH, 65% to 100% EtOH, 70% to 100% EtOH, 75% to 100% EtOH, 80% to 100% EtOH, 85% to 100% EtOH, 90% to 100% EtOH, 95% to 100% EtOH, and all ranges and sub-ranges therebetween.

[0141] In a specific embodiment, the RNA-bound beads are washed with ETOH two or more times, preferably three times, more preferably four times.

[0142] The beads were homogenized by stirring at 1000 rpm for one minute. A magnetic field was applied for at least 120 seconds and the supernatant was removed.

[0143] RNA was eluted by adding elution buffer with 1 mM sodium citrate at pH 6.4. Incubate with elution buffer for at least 5 minutes while stirring the sample at 800 rpm at room temperature. The supernatant was then removed and recovered into an RNAse-free tube. The elution procedure was repeated once more and the two eluted samples were finally pooled together.

[0144] Example 3

[0145] DNA purification

[0146] Mix 50ul of PCR product containing DNA with 35μL of nuclease-free water and 100μL of binding buffer (containing 4M NaCl, 10mM Tris-HCl, 1mM EDTA, 30% PEG 8000, pH 8.0) to make a total volume of 185ul DNA sample solution. Add 15μL of magnetic beads to the resulting DNA solution and vortex for at least 3 minutes. In order to properly bind, incubate the magnetic beads and DNA mixture at room temperature on a tilt rotor for at least 2 minutes.

[0147] In a subsequent step, the beads are placed in a magnetic field for up to 3 minutes, and then the supernatant is removed. In the next step, the DNA-bound beads are washed three times with 700 μL of wash buffer (containing 10 mM Tris-HCl, 1 mM EDTA, 70% EtOH). The beads are homogenized by shaking for 3 minutes and incubated on a tilt rotor for 2 minutes. A magnetic field is applied for 3 minutes, and then the supernatant is removed. The tube containing the DNA-bound beads is placed on a magnetic stand for 10 minutes to air-dry the precipitate of the DNA-bound beads.

[0148] DNA was eluted by adding 10 μL of 5 mM Tris / HCl, pH 8.0 elution buffer.

[0149] 3. Resuspend the DNA-bound beads in elution buffer by agitating at 800 rpm for 5 minutes and then incubating on a magnetic stand for 3 minutes.

[0150] The supernatant containing the DNA was then collected and transferred to a fresh tube.

[0151] Similar results were obtained when the binding buffer contained 10% to 40% v / v EtOH instead of PEG.

[0152] Example 4

[0153] Nucleic acid purification process

[0154] In another example of nucleic acid purification, the binding and subsequent magnetic separation steps are repeated up to 10 times before a washing step (hereinafter referred to as sequential binding steps). Figure 3 A schematic diagram of such a process is given.

[0155] As a comparative study, a method involving consecutive binding steps (a process such as Figure 3 as shown) and with a one-round combination method (this process is as Figure 2 The yield (amount of purified nucleic acid) and amount of buffer used for each method were compared.

[0156] Buffer:

[0157] The buffer compositions used are as described in Example 1. In the binding buffer, the proportion of EtOH can be 13% to 100%, and in the washing buffer, the proportion of EtOH can be 50% to 100%.

[0158] method:

[0159] The coated silica magnetic beads, the IVT sample with nucleic acid and the binding buffer are mixed together. The resulting mixture is incubated to allow the nucleic acid to reversibly bind to the beads. Subsequently, a magnetic field is applied to separate the magnetic particles with bound nucleic acid from the liquid phase, which is discarded or retained for other uses. These consecutive binding steps are repeated up to 10 times. It was found that this repetition significantly reduced the use of wash buffer and elution buffer, thereby significantly reducing the amount of organic solvent (e.g., EtOH) required.

[0160] These sequential binding steps were performed using two different methods.

[0161] These methods are:

[0162] (i) In each cycle, add the magnetic beads + nucleic acid to the magnetic beads in the previous step, or

[0163] (ii) Place all the beads in the tube from the beginning (first round) and add nucleic acids sequentially in each cycle.

[0164] Both consecutive binding steps produced similar results. After the binding step, up to four consecutive washing steps were performed using a washing buffer. In some examples, the number of washing steps was reduced to one or more than two. After the washing steps, the sample was eluted. In order to increase the yield of the collected nucleic acid, the elution step was repeated.

[0165] result:

[0166] The results show that for the same amount of nucleic acid, the process described in this example comprising 10 cumulative binding reactions reduces the consumption of organic solvent per unit of purified mRNA by 82%.

[0167] Table 1 shows the use of ethanol in the purification of 38.4 L IVT in two embodiments of the present invention.

[0168] Table 1:

[0169]

[0170] The present invention is not limited to the embodiments described in the examples and / or shown in the figures. On the contrary, the method of the present invention can be implemented in many different ways without departing from the present invention.

[0171] Example 5: Quality maintenance of purified nucleic acids in binding buffers at various salt concentrations and pH

[0172] The method was performed as described in Example 2 except that different binding buffer compositions and / or pH were used to purify the RNA obtained from the IVT reaction.

[0173] Experiments were performed using three different NaCl concentrations in the final binding reaction composition: 0.4 (A), 1.0 M (B) and 1.6 M (C) NaCl and at three different pHs (6.9, 7.9 and 9.0).

[0174] The quality and functional activity of purified RNA were assessed by determining the protein / RNA ratio, the amount of target DNA extracted from the bulk purified RNA, and the capping efficiency of individual purified RNA samples.

[0175] RNA / Protein Ratio:

[0176] At the tested salt concentrations and / or pH of the binding buffer, the purification quality was maintained (see Table 2 below). The technical effects in the pH and salt ranges of the binding buffer were determined according to the publication.

[0177] For each purified RNA sample, the protein content (using μBCA (micro BiCinchoninic acid Assay)) and RNA content (using Nanodrop) were measured. Figure 4A As shown, the results indicate that there is no significant correlation between the salt concentration of the binding buffer and the protein / RNA ratio. Figure 4B Similar results were obtained under different pH conditions: no significant correlation between the tested pH of the binding buffer and the protein / RNA ratio was detected (see also Table 2 below).

[0178] Table 2

[0179]

[0180] rDNA content:

[0181] Another quality metric for purified RNA is the amount of residual DNA (rDNA) in the bulk of the purified RNA. The amount of rDNA in each purified RNA sample was measured using ELISA and the results were compared. The results showed that the methods disclosed herein performed well at the tested salt concentrations of the binding buffer ( Figure 5A ) and pH( Figure 5B ) conditions were equally effective in removing DNA from RNA samples because the variables tested had no effect on the amount of target DNA extracted from the purified RNA.

[0182] Capping efficiency:

[0183] The capping efficiency of RNA purified using different binding buffers of the present invention was evaluated by HPLC. The results showed that the capping efficiency of the purified RNA samples was similar when different binding buffers were used (see Table 3 below).

[0184]

[0185] In summary, the results show that the methods of the present disclosure are successful in purifying nucleic acids when using a wide range of binding buffers with salt concentrations and / or pH. The results clearly show that the tested range of sodium chloride concentrations and / or pH in the binding buffer are effective in purifying RNA and maintaining quality requirements such as RNA / protein ratio, rDNA content, and RNA capping efficiency.

Claims

1. A method for purifying nucleic acid, comprising: a) combining a sample comprising at least one target nucleic acid in a binding buffer at a pH of 5 to 10 with silica-based magnetic particles to form a solution; Wherein, the binding buffer comprises at least one salt with a concentration of 0.1M to 5M, at least one first alcohol with a concentration of 10% to 50% v / v and / or polyethylene glycol (PEG) with a concentration of 10% to 40% (v / v), b) incubating the solution for a sufficient time to allow the at least one nucleic acid to reversibly bind to the magnetic particles to form modified magnetic particles; c) separating the modified magnetic particles from the combined solution by applying a magnetic field; d) washing the at least one modified magnetic particle with at least one wash buffer comprising at least one second alcohol in a concentration of 60% to 100%, e) combining the modified magnetic particles with an elution buffer to elute the target nucleic acid from the magnetic particles, wherein the pH of the elution buffer is about 5 to 10, and wherein the conductivity of the elution buffer is 0.001 mS to 40 mS / cm, or the total salt concentration is 0 mM to 50 mM.

2. The method according to claim 1, wherein: Steps a to c are repeated one or more times, and then step d is performed.

3. The method according to claim 2, wherein: Repeat steps a to c 1 to 10 times.

4. The method according to any of the preceding claims, adding another sample comprising the at least one target nucleic acid in a binding buffer and optionally unmodified silica-based magnetic particles to the modified magnetic particles of the preceding step c to form a solution, and repeating steps b and c.

5. A method according to any one of the preceding claims, wherein: The washing in step d is repeated one or more times, wherein after each washing step, the washing buffer is removed from the modified beads.

6. A method according to any one of the preceding claims, wherein: The elution is repeated one or more times in succession.

7. A method according to any one of the preceding claims, wherein: The concentration of the at least first alcohol is 15% to 20% v / v.

8. A method according to any one of the preceding claims, wherein: The salt of the binding buffer is NaCl, wherein the concentration of NaCl is preferably 0.5M to 2M.

9. A method according to any one of the preceding claims, wherein: The binding buffer does not contain guanidine salts.

10. A method according to any one of the preceding claims, wherein: The first alcohol in the binding buffer is selected from isopropanol, methanol, ethanol, butanol and / or a combination thereof.

11. A method according to any one of the preceding claims, wherein: The wash buffer comprises 60% to 80% by volume of at least one second alcohol, wherein the at least one second alcohol is selected from isopropanol, methanol, ethanol, butanol, or a combination thereof.

12. A method according to any one of the preceding claims, wherein: The elution buffer is selected from water or at least one pH buffer having a concentration of 8 mM to 12 mM, and / or at least one ion chelator having a concentration of 0.8 mM to 1.2 mM.

13. A method according to any one of the preceding claims, wherein: The elution buffer was sodium citrate.

14. A method according to any one of the preceding claims, wherein: The elution buffer does not contain guanidine salts.

15. A method according to any one of the preceding claims, wherein: The target nucleic acid is DNA or RNA, more preferably mRNA obtained by in vitro transcription (IVT) reaction.

16. A method according to any one of the preceding claims, wherein: The at least one magnetic particle is coated with one or more ligands that interact with the nucleic acid.

17. A method according to any one of the preceding claims, wherein: The method is semi-automatic or fully automatic.

18. A method according to any one of the preceding claims, wherein: The at least one magnetic particle is present in the binding buffer at a concentration of 0.1 μg / μl to 200 μg / μl.

19. The method according to claim 1, wherein: The volume ratio of the sample comprising at least one nucleic acid to the binding buffer is 1:1 to 1:

3.

20. A nucleic acid purification kit comprising: a) a binding buffer having: a pH of 5 to 10; at least one salt, which is NaCl, having a concentration of 0.1 M to 5 M; ethanol at a concentration of 10% to 50% volume / volume; and / or PEG at a concentration of 10% to 40%; b) a wash buffer comprising at least 60% by volume ethanol; c) an elution buffer selected from water, a solution comprising at least one buffer compound, the conductivity of which is between 0.001 mS and 40 mS / cm, or wherein the total salt concentration is between 0 mM and 50 mM; d) A solution comprising magnetic particles.

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