WO2025003501A1

WO2025003501A1 - Nucleic acid preparation

Info

Publication number: WO2025003501A1
Application number: PCT/EP2024/068402
Authority: WO
Inventors: Hannah KIM LINDSTRÖM; Frauke Margarete HENJES; Voin PETROVIĆ
Original assignee: Life Technologies AS
Current assignee: Life Technologies AS
Priority date: 2023-06-30
Filing date: 2024-06-28
Publication date: 2025-01-02
Anticipated expiration: 2025-12-30
Also published as: GB202310075D0; CN121620591A

Abstract

Provided herein is an aqueous nucleic acid purification buffer comprising a polar aprotic solvent. Also provided is use of such a purification buffer to precipitate a nucleic acid from solution to a solid support, thereby providing a nucleic acid bound solid support. A method of processing a nucleic acid is also provided, comprising exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support. Further provided are kits and compositions comprising such an aqueous nucleic acid purification buffer, as well as related nucleic acid analysis apparatus, and use of said kits.

Description

Nucleic Acid Preparation

[0001] This invention relates to procedures for preparation of nucleic acids, as well as buffers, kits, compositions and apparatus relating to the same. The methods may involve precipitation of nucleic acids from solution to a solid support, with optional washing of the nucleic acid bound solid support and release of the nucleic acid from the washed solid support. Also provided are upstream processes such as lysis (e.g. to release nucleic acids from biological samples into solution), and/or downstream processes, such as amplification, detection, analysis and the like.

BACKGROUND

[0002] The isolation of nucleic acids, such as DNA or RNA, is an important step in many molecular biology, medical and diagnostic procedures. Indeed the separation of nucleic acids from the complex mixtures in which they are often found is frequently necessary before other studies and procedures can be performed on nucleic acids. For example, many types of nucleic acid detection, cloning, sequencing, amplification, hybridisation, cDNA synthesis, studying nucleic acid structure and composition require relatively clean samples of the nucleic acids before the relevant experiments can be performed. The presence of large amounts of cellular or other contaminating material e.g. proteins or carbohydrates, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. In addition, DNA may contaminate RNA preparations and vice versa. Thus, methods for the isolation of nucleic acids from complex mixtures such as cells, tissues etc. are demanded, not only from the preparative point of view, but also in many methods which rely on the identification of DNA or RNA e.g. diagnosis of microbial or viral infections, forensic science, tissue and blood typing, genotyping, detection of genetic variations etc. The purification of DNA or RNA from more enriched but still contaminated samples is also desirable, e.g. to purify synthetically prepared nucleic acid material, e.g. to purify PCR products from contaminating salts, excess primers and/or dNTPs.

[0003] A number of methods are known for the isolation of nucleic acids, but these often rely on a complex series of extraction and washing steps and are therefore time consuming and laborious to perform. Moreover, the use of materials such as alcohols and other organic solvents, chaotropes and proteinases is often involved. This is disadvantageous since such materials tend to interfere with enzymatic reactions and other downstream processing applications.

[0004] In particular, typical methods for the isolation of nucleic acids from complex starting materials such as blood or blood products or tissues involve lysis of the biological material by a detergent or chaotrope, possibly in the presence of a protein degrading enzyme, followed by several extractions with organic solvents e.g. phenol and/or chloroform, ethanol precipitation, centrifugations and dialysis of the nucleic acids. The purification of RNA from DNA may involve a selective precipitation with LiCI or a selective isolation with guanidinium thiocyanate combined with phenol extractions and ethanol precipitation. Not only are such methods cumbersome and time consuming to perform, but the relatively large number of steps required increases the risk of degradation, sample loss or cross-contamination of samples where several samples are simultaneously processed. In addition, the reagents used in such purifications often provide a number of other disadvantages, for example flammability, high viscosity or may be subject to environmental, health or safety (EHS) concerns.

[0005] Methods have been proposed which rely upon the use of a solid phase. US 5, 234, 809, for example, describes a method where nucleic acids are bound to a solid phase in the form of silica particles, in the presence of a chaotropic agent such as a guanidinium salt, and thereby separated from the remainder of the sample. WO 91/12079 describes a method whereby nucleic acid is trapped on the surface of a solid phase by precipitation. Generally speaking, monohydric alcohols (ethanol or isopropanol) and salts are used as precipitants.

[0006] Ethanol and/or isopropanol when included in sufficient quantity in an aqueous medium act to precipitate dissolved nucleic acids out of solution and to keep out of solution nucleic acids already in the extra-solution phase. Ethanol and isopropanol may therefore be described as "anti-solvating" agents for nucleic acids. Without being bound by theory, it is believed that the ethanol or isopropanol reduce the amount of free water available to dissolve the nucleic acid. These two monohydric alcohols are therefore used during the processing of nucleic acids to precipitate them (e.g. bind them to a solid support) and/or to keep them from dissolving in the aqueous medium, in the latter case while other procedures are carried out. In any particular workflow, one or a combination of these alcohols may be used to precipitate nucleic acid, to keep nucleic acid precipitated, or both to precipitate nucleic acid and to keep it precipitated.

[0007] US 5,705,628 and US 5,898,071 describe methods of isolating nucleic acid fragments using a combination of large molecular weight polyalkylene glycols (e.g. polyethylene glycols) at concentrations of from 7 to 13% with salt in the range of 0.5 to 5M to achieve binding to functional groups on a solid support which acts as a bioaffinity absorbent for DNA. WO 2012/069660 A1 describes methods of isolating nucleic acids onto solid supports using polyols. EP3215620B1 describes chaotrope-free and volatile free nucleic acid extraction from plasma.

[0008] Although such methods generally speed up the nucleic acid separation process, there are disadvantages associated with basing procedures involving solid phase nucleic acids on current agents for inducing and/or maintaining the precipitated state, for example alcohols (such as ethanol or isopropanol), chaotropes, salts and large molecular weight molecules (such as polyethylene glycols). [0009] Large molecular weight molecules increase the viscosity of the liquid which reduces the efficiency with which purification protocols can be conducted. In the case of separation of magnetic beads, such large molecules reduce the speed of isolation as the time of contact with the magnet to separate the beads has to be increased. Furthermore, the removal of supernatant in such systems is more difficult in the presence of the large molecular weight molecules. Polyols perform better than large molecular weight molecules, however they still tend to have a higher than ideal viscosity which may affect accuracy of liquid transfer operations, especially when used in Point-of-Care (POC) or microfluidic devices with narrow channels and may result in reduced removal of impurities.

[0010] Chaotropes need to be used at high molarity, resulting in viscous solutions which may be difficult to work with, especially in RNA work. Amplification procedures such as PCR, and other enzyme-based reactions, are very sensitive to the inhibitory or otherwise interfering effects of alcohols and other agents. Moreover, the drying of the nucleic acid pellet which is necessary following alcohol (i.e. ethanol or isopropanol) precipitation and the problems with dissolving nucleic acids, are also known to lead to artefacts in enzyme-based procedures such as amplification reactions. Furthermore, liquids with a high content of ethanol or isopropanol are volatile and flammable, which increases transport and disposal costs for the liquids and hence increases the cost of the overall process.

[0011] There is a need for improved methods of nucleic acid isolation and for improved reagents for use in such methods. There is a particular desire for EHS-friendly methods which are quick and simple to perform, which enable good yields to be obtained with minimal losses, and which avoid (or at least reduce) the use of volatile solvents and chaotropic agents or alcohol precipitation or the use of high levels of salt and/or high molecular weight compounds with high viscosity and preferably be compatible for adaptation in point-of-care and near point- of-care applications.

BRIEF SUMMARY OF THE DISCLOSURE

[0012] This invention is based in part on an understanding that there are polar aprotic solvents that may be utilized in buffers for purification of nucleic acids, instead of the alcohols, polyols, polyethylene glycols or similar compounds used previously. This may provide a number of advantages, such as low viscosity for improved handling I ease of separation from any solid phase, and/or low flammability for improved safety, and/or good compatibility with downstream processes (such as PCR amplification or mass spectrometric analysis).

[0013] A first aspect of the invention provides an aqueous nucleic acid purification buffer comprising a polar aprotic solvent.

[0014] The polar aprotic solvent may be present in an amount of at least about 2 %wt. The polar aprotic solvent may be present in an amount of not more than about 80 %wt.

For example, the polar aprotic solvent may be present in an amount of from between about 4 %wt to about 75 %wt.

[0015] The polar aprotic solvent may have a boiling point of more than 100 °C at 1 atm. For example, the polar aprotic solvent may have a boiling point of more than 150°C at 1 atm.

[0016] The polar aprotic solvent may comprise 2, 3 or 4 heteroatoms selected from O and N. For example, the polar aprotic solvent may comprise 2 or 3 heteroatoms selected from O and N. The polar aprotic solvent may comprise 2 or 3 O heteroatoms. The polar aprotic solvent may comprise 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. The polar aprotic solvent may comprise 2 O heteroatoms. The polar aprotic solvent may comprise 3 O heteroatoms. The polar aprotic solvent may comprise 1 O heteroatoms and 1 or 2 N heteroatoms. The O heteroatoms may comprise =0 and/or -O-.

[0017] The polar aprotic solvent may comprise 4, 5, 6, 7, 8, 9 or 10 carbon atoms. For example, the polar aprotic solvent may comprise 5, 6, 7, or 8 carbon atoms; e.g. the polar aprotic solvent may comprise 6 or 8 carbon atoms. The polar aprotic solvent may comprise 5 carbon atoms. The polar aprotic solvent may comprise 6 carbon atoms. The polar aprotic solvent may comprise 7 carbon atoms. The polar aprotic solvent may comprise 8 carbon atoms.

[0018] The polar aprotic solvent may have a viscosity of less than 50 cP at 20 °C and 1 atm. For example, the polar aprotic solvent may have a viscosity of less than 40 cP or 30 cP at 20 °C and 1 atm; e.g. the polar aprotic solvent may have a viscosity of less than 25 cP at 20 °C and 1 atm.

[0019] The polar aprotic solvent may have a flash point of at least 50°C.

[0020] The polar aprotic solvent may be liquid at 0 °C and 1 atm.

[0021] The polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N- Formylmorpholine. For example, the polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, and dipropylene glycol dimethyl ether.

[0022] The purification buffer may further comprise one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof. For example, the purification buffer may comprise two or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent; e.g. the purification buffer may comprise three or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent.

[0023] The purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of an alcohol or a polyol. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of alcohol or polyol may be preferred.

[0024] The purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of a polyethylene glycol PEG, a TEG, or an LPA. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of a PEG, a TEG, or an LPA may be preferred.

[0025] The purification buffer may be a binding buffer and/or a wash buffer. For example, the purification buffer may be a binding buffer and a wash buffer. The purification buffer may be a binding buffer. The purification buffer may be a wash buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support.

[0026] In view of this, in some embodiments the wash buffer may represent an aqueous dilution of the binding buffer. The aqueous dilution may also comprise other reagents that may not be present in the binding buffer, such as: a buffering agent, or a buffering agent and an inorganic salt. The wash buffer may comprise a 1.1 to 5 times aqueous dilution of the binding buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the binding buffer.

[0027] In embodiments, a workflow or kit could comprise both a binding buffer of the present invention and a wash buffer of the present invention, where the binding buffer and wash buffers have different compositions, i.e. the wash buffer is neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.

[0028] In embodiments, the purification buffer does not contain ethanol, isopropanol or 2- methyl-1,3- propanediol. In embodiments, the purification buffer does not contain dimethylsulfoxide (DMSO). In embodiments, the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or DMSO. In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol). In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol) or DMSO. In embodiments, the purification buffer does not contain a salt or a chaotrope.

[0029] A second aspect of the invention provides the use of a purification buffer of the first aspect, to precipitate a nucleic acid from solution to a solid support, thereby providing a nucleic acid bound solid support.

[0030] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties.

[0031] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support may be a component of a robotic liquid handling platform.

[0032] The use may further comprise washing the nucleic acid bound solid support with a wash buffer. The wash buffer may be or comprise the purification buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding. For example, the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.

[0033] The use may further comprise contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support. The elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof.

[0034] The separated nucleic acid may be further subjected to one or more additional processes. The one or more additional processes may be selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

[0035] The amplification may comprise PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. The isothermal amplification may comprise loop- mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicasedependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA) (for an overview see “Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", Infection and Drug Resistance 2020:13, 455-483 and references therein). [0036] The sequencing may comprise next generation sequencing. “Next-generation sequencing” and “high-throughput sequencing” are sequencing techniques that parallelize the sequencing process, producing a high number of sequences at once. The nextgeneration sequencing methods may comprise single molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 Life Sciences), sequencing by ligation (e.g., SOLiD sequencing of Life Technologies), sequencing by synthesis and reversible terminator (e.g., Illumina), reversible dye terminator-based sequencing (e.g. Solexa), Oxford Nanopore sequencing, FRET-donor-polymerase-based sequencing (VisiGen Biotechnologies), extension-based single molecule sequencing (Helicos Bioscience), hybridization sequencing, nucleic acid imaging technologies such as transmission electron microscopy, and the like.

[0037] The nucleic acid may be DNA. The DNA may, for example, be one or more of synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA.

[0038] The nucleic acid may be RNA. The RNA may, for example, be one or more of mRNA (e.g. isolated from a biological sample or in v/fro-transcribed), siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).

[0039] A third aspect provides a method for processing a nucleic acid. The method comprises exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support.

[0040] In preferred embodiments, the aqueous medium comprising the polar aprotic solvent is an aqueous nucleic acid purification buffer as defined in the first aspect.

[0041] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties.

[0042] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic.

[0043] The method may further comprise washing the nucleic acid bound solid support with a wash buffer. The wash buffer may be or comprise the purification buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding. For example, the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.

[0044] The method may further comprise contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid. The elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof.

[0045] The method may further comprise subjecting the nucleic acid (e.g. the separated nucleic acid) to one or more additional processes. The one or more additional processes may be selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, blotting, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation, and genome editing.

[0046] The amplification may comprise PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. The isothermal amplification may comprise loop- mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicasedependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA) (for an overview see “Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", Infection and Drug Resistance 2020:13, 455-483 and references therein).

[0047] The sequencing may comprise next generation sequencing. “Next-generation sequencing” and “high-throughput sequencing” are sequencing techniques that parallelize the sequencing process, producing a high number of sequences at once. The nextgeneration sequencing methods may comprise single molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 Life Sciences), sequencing by ligation (e.g., SOLiD sequencing of Life Technologies), sequencing by synthesis and reversible terminator (e.g., Illumina), reversible dye terminator-based sequencing (e.g. Solexa), Oxford Nanopore sequencing, FRET-donor-polymerase-based sequencing (VisiGen Biotechnologies), extension-based single molecule sequencing (Helicos Bioscience), hybridization sequencing, nucleic acid imaging technologies such as transmission electron microscopy, and the like.

[0048] The sample may comprise or be a pre-treated or untreated biological sample, clinical environmental sample, or an enzymatic reaction mixture. The sample may be or comprise a pre-treated or untreated biological sample. The biological sample may be in a physiological buffer or transport medium. The biological sample may be a harvested or biopsied sample or a cultured sample. The sample may be or comprise an environmental sample. The sample may be or comprise an enzymatic reaction mixture.

[0049] The biological sample may be any suitable biological sample. Exemplary biological samples may comprise one or more of blood, blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin-fixed paraffin- embedded (FFPE) sample, biopsy sample, bone marrow or other tissue sample, plant sample, cell lysate, bacterial or yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, exhalates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk. In some instances, the biological sample may be a clinical sample. In some instances, the sample may be a cell-free sample.

[0050] The environmental sample may comprise a water sample (such as a wastewater sample), a soil sample, a sediment sample, a surface swab, an air derived sample (such as an air filter residue), a cosmetic, a food ingredient or a food sample, or a combination thereof.

[0051] The enzymatic reaction mixture could be any such mixture that may contain nucleic acid sequences. For example, the enzymatic reaction mixture may comprise in vitro transcription reaction mixture, a reverse transcription reaction mixture, a second strand synthesis reaction mixture, a nucleic acid assembly reaction mixture, an amplification reaction mixture, a library preparation reaction mixture, a restriction reaction mixture, a nucleic acid assembly reaction mixture, or a barcoding reaction mixture.

[0052] Processes of the present invention can be used for extraction and analysis of fragmented DNA. DNA can be fragmented by a variety of methods known in the art, such as nuclease digestion (including digestion with restriction endonucleases and DNases), sonication, heat, mechanical disruption (such as by shearing or vortexing), and chemical treatment. Applicable chemical treatments include, for example, use of metal ions such as iron (Zhang et al., Nucl. Acids Res. 29(13):e66, 2001), oxidizing agents such as bisulfite (Ehrich et al., Nucl. Acids Res. 35(5):e29, 2007), and antibiotics and drugs such as bleomycin (Chen et al., Nucl. Acids Res. 3<5(l l):3781-3790, 2008). A preparation of fragmented DNA can contain fragments of a range of sizes or may be relatively limited in size range. Those skilled in the art will recognize that the actual size of fragments will be determined by such factors as the fragmentation method selected and the conditions used (e.g., time of treatment).

[0053] In embodiments, the aqueous medium does not contain ethanol, isopropanol or 2- methyl-1,3- propanediol. In embodiments, the aqueous medium does not contain dimethylsulfoxide (DMSO). In embodiments, the aqueous medium does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or DMSO. In embodiments, the aqueous medium does not contain an alcohol (including any diol or polyol). In embodiments, the aqueous medium does not contain an alcohol (including any diol or polyol) or DMSO. In embodiments, the aqueous medium does not contain a salt or a chaotrope.

[0054] The method may further comprise performing point-of-care detection. The point- of-care detection may comprise pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection. The pathogen detection or biological warfare agent detection may comprise viral, bacterial, single-celled fungi or protozoan nucleic acid detection.

[0055] A fourth aspect provides a kit comprising an aqueous nucleic acid purification buffer of the first aspect, and a solid support.

[0056] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties.

[0057] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc or a slide. The solid support may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support may be a component of a robotic liquid handling platform.

[0058] The kit may comprise one or more of a lysis or lysis/binding buffer, a wash buffer, and an elution buffer. The lysis buffer and/or the wash buffer may comprise the polar aprotic solvent of the aqueous nucleic acid purification buffer.

[0059] A fifth aspect provides a composition of matter. The composition of matter comprises (i) a solution comprising an aqueous nucleic acid purification buffer in accordance with the first aspect; and (ii) a nucleic acid solid support.

[0060] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties.

[0061] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic.

[0062] The nucleic acid may be DNA; such as plasmid DNA, cDNA, cfDNA and I or genomic DNA. The nucleic acid may be RNA; such as one or more of mRNA (e.g. isolated from a biological sample or in v/tro-transcribed), siRNA, microRNA, tRNA, or cfRNA.

[0063] A sixth aspect provides use of a kit of fourth aspect in an automated nucleic acid analysis platform.

[0064] A seventh aspect provides a nucleic acid analysis apparatus. The apparatus comprises an automated nucleic acid analysis platform and a kit of the fourth aspect. The automated nucleic acid analysis platform comprises a portion configured to house the solid support.

[0065] The automated nucleic acid analysis platform may be a point-of care assay instrument. The point-of-care assay instrument may be adapted for pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection. The pathogen detection or biological warfare agent detection may comprise viral, bacterial, single-celled fungi or protozoan nucleic acid detection.

[0066] The present invention has multiple applications in laboratory research, human and veterinary medicine, public health and sanitation, forensics, anthropological studies, environmental monitoring, and industry. Such applications include, without limitation, bacterial and viral detection and typing, microbial drug resistance screening, viral load assays, genotyping, infection control and pathogen screening (of, e.g., blood, tissue, food, cosmetics, water, soil, and air), pharmacogenomics, detection of cell-free DNA in plasma, white cell counting, and other fields where preparation and analysis of DNA from biological samples is of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 provides an outline of exemplary workflows for isolation of nucleic acids from a liquid sample. (A) provides a general schematic, (B) provides an example of a standard workflow for isolation of nucleic acids that uses isopropanol (I PA) in the binding buffer, and (C) provides an exemplary workflow of the invention, which uses N- butylpyrrolidin-2-one (TamiSolve™) in the binding buffer. As the skilled person will appreciate, exemplary workflow (C) may be adapted to use other binding and wash buffers as disclosed herein. Figure 2 illustrates the DynaMag™- 2 magnet, SKU 12321 D.

Figure 3 illustrates the recovery of nucleic acids from silanol functionalised stationary phases using aprotic solvents selected from dihydrolevoglucosenone (Cyrene™), dipropylene glycol dimethyl ether (Proglyde™), ZV-butylpyrrolidin-2-one (TamiSolve™), or N-formylmorpholine (N-FM) in the binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. (A) indicates the MS2 RNA recovery on silane beads, while (B) indicates the M 13 DNA recovery on silane beads.

Figure 4 illustrates the recovery of nucleic acids from carboxylic acid functionalised stationary phases using aprotic solvents selected from Cyrene™, Proglyde™, TamiSolve™, or N-Formylmorpholine (N-FM) in the binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. (A) indicates the MS2 RNA recovery on carboxy beads, while (B) indicates the M13 DNA recovery on carboxy beads.

Figure 5 illustrates PCR results for nucleic acids recovered from carboxylic acid functionalised stationary phases for 8 different extractions, including a fully aprotic solvent workflow. (A) shows results for RNA. (B) shows results for DNA.

Figure 6 provides the calculated percent recovery, based on the number of nucleic acid copies that were spiked into the samples. (A) shows results for RNA. (B) shows results for DNA.

DETAILED DESCRIPTION

[0068] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0069] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0070] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0071] For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading “Background” is relevant to the invention and is to be read as part of the disclosure of the invention.

[0072] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Definitions

[0073] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0074] The term “nucleic acid” means, unless otherwise stated, a polynucleotide molecule made up of ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions, i.e. "hybridisation" or the formation of a "duplex". Thus, the nucleic acid may be DNA or RNA or any modification thereof, including conformationally restricted or nucleobase analogue-bearing oligomers such as “locked-nucleic acids” (LNA) or “peptide nucleic acids” (PNA) or other derivatives containing non-nucleotide backbones. The nucleic acid may be a naturally occurring molecule, i.e. DNA or RNA but also include DNA/RNA hybrids where the DNA is in separate strands or in the same strand) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester linkage to the 5' position of the pentose of the next nucleotide. Nucleic acids used in various embodiments may comprise chemically, enzymatically, or metabolically modified forms of nucleotides or combinations thereof, such as primers, probes, oligonucleotides or aptamers. Exemplary types of DNA include synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA. Exemplary types of RNA include mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).

[0075] The term “purification buffer” means, unless the context requires otherwise, a buffer that is useful for precipitating a nucleic acid from solution to a solid support and/or a buffer that is useful for washing a nucleic acid bound or precipitated to a solid support. Exemplary purification buffers may therefore also be considered binding buffers or washing buffers.

[0076] The term “binding buffer” means, unless the context requires otherwise, a buffer that is useful for precipitating a nucleic acid from solution to a solid support. Nucleic acids are often solvated in aqueous solutions, therefore typical binding buffers are miscible in aqueous solution, e.g. a binding buffer may be provided as an aqueous buffer. Exemplary binding buffers of the present invention are aqueous buffers comprising a polar aprotic solvent.

[0077] The terms “wash buffer” or “washing buffer” mean, unless the context requires otherwise, a buffer that is useful for washing a nucleic acid bound to a solid support. Binding buffers, in view of their function to precipitate nucleic acid from solution, may also be useful as wash buffers, as they should provide minimal loss of the nucleic acid from the solid support during washing. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the binding buffer, such as a 1.1 to 5 times dilution of a corresponding binding buffer.

[0078] The term “solid support” or “solid phase” means, unless otherwise stated, a material that is substantially insoluble in a selected solvent system (e.g. comprising an aqueous buffer), or which can be readily separated (e.g., by precipitation) from a selected solvent system in which it is soluble. In the present disclosure, solid supports that are substantially insoluble in a selected solvent system (e.g. comprising an aqueous buffer) may be preferred. Such solid supports are not limited to a specific type of support, and a large number of such solid supports are available and are known to one of ordinary skill in the art. Exemplary solid supports include, but are not limited to, solid and semi-solid matrixes, such as aerogels and hydrogels, resins, particles, beads (including magnetic beads, such as coated magnetic beads), biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtiter plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports. Where the solid support comprises beads, monodisperse (magnetic or non-magnetic) beads may be preferred, as monodisperse beads may provide more consistent performance in assays. A solid support may comprise magnetic beads. Solid supports useful in the practice of the present invention have a hydrophilic surface that allows binding of nucleic acids, e.g. by non-covalent interactions. The hydrophilic surface of the solid support may comprise a charged surface in the pH range of about 6 to about 8.

[0079] The term “nucleic acid solid support” means, unless otherwise stated, a solid support or solid phase as disclosed herein that is adapted for binding of nucleic acids, e.g. by non-covalent interactions. A nucleic acid solid support may have a hydrophilic surface that allows binding of nucleic acids. The hydrophilic surface of the solid support may comprise a charged surface in the pH range of about 6 to about 8.

[0080] The term “monodisperse” means that for a plurality of particles or beads (e.g. at least 100, more preferably at least 1 ,000) the particles or beads have a coefficient of variation (CV) or % polydispersity of their diameters of less than 20%, for example less than 15%, typically of less than 10% and optionally of less than 8%, e.g. less than 5%. The term monodisperse is used herein to characterize a population of particles or beads with low heterogeneity and a homogenous size distribution. The size distribution of a particle or bead may be defined by the percentage CV (coefficient of variation) which may be determined on a CPS disc centrifuge as described e.g. in the Analytical Methods section of WO2017211913A1 which is incorporated by reference herein. CV is defined as 100 times (standard deviation) divided by average where “average” is mean particle or bead diameter 10 and standard deviation is standard deviation in particle size. The CV for a plurality of particles may for example be within a range of 50 to 100%. For example, a monodisperse particle or beads population may have more than 90%, preferably more than 95% of the particles or beads with sizes within their mean diameter of ± 5 %.

[0081] The term “surface”, in relation to a solid support means, unless otherwise stated, a solvent accessible part of the solid support. This includes any outer surface of the solid support, as well as the surface of solvent accessible pores of a porous solid support. The surface of the solid support may comprise surface hydroxyl groups. The surface hydroxyl groups may comprise part of silanol, carboxyl, or saccharide moieties. The surface of the solid support may comprise negatively charged groups (such as acidic groups) and/or the surface of the solid support may comprise positively charged groups. The surface of the solid support may comprise acidic groups. The surface of the solid support may comprise carboxyl groups. The surface of the solid support may comprise positively charged groups, for example one or more positively ionizable groups with a pK_a of between about 5 and 8 (e.g. with a pK_a of between about 6 and 7). The surface of the solid support may comprise polyethylene imine groups, morpholine groups, alanine groups, polyhydroxy amine groups (such as Tris, Bis-Tris, and the like). The hydrophilic surface of the solid support may comprise biological buffer covalently bound thereto; for example a biological buffer as described in US 6,914,137 B2, column 5, line 55 to column 6, line 59, covalently bound as described in said document at column 7, lines 29 to 64, the content of which is incorporated herein by reference in its entirety.

[0082] The term “alcohol” means, unless otherwise stated, a compound comprising a hydroxyl group, for example an alkane or alkene substituted by a hydroxyl group. Exemplary alcohols include Ci-Ce alcohol; such as methanol, ethanol, propanol, isopropanol, or butanol. In some embodiments described herein, purification buffers, binding buffers, wash buffers and aqueous medium that comprise an aprotic solvent may not comprise alcohol.

[0083] The term “polyol” means, unless otherwise stated, a compound comprising multiple (e.g. 2) hydroxyl groups. Exemplary polyols include, but are not limited to, a C₂- C10 polyol, where the polyol can be an alkane or alkene substituted by at least two hydroxyl groups and optionally interrupted by 1, 2 or 3 ether linkages. Particular examples of polyols include 2-methyl-1,3-propanediol, tripropylene glycol, and butanediol. In some embodiments described herein, purification buffers, binding buffers, wash buffers and aqueous medium that comprise an aprotic solvent may not comprise polyol.

[0084] The term “magnetic” means responds to a magnetic field. For example, magnetic beads respond to a magnetic field. Magnetic materials (such as magnetic beads) may be paramagnetic or superparamagnetic. When the magnetic material is paramagnetic, the magnetic properties are switched off when the magnetic field is removed. When the magnetic material is superparamagnetic, the magnetic material becomes saturated at relatively low magnetic fields and switching off of the magnetic properties with removal of the magnetic field is very rapid/instant. Some magnetic material, e.g. iron oxides, form superparamagnetic crystals when the size of the crystals is sufficiently small (e.g. below about 15 nm scale for iron oxides).

[0085] The term “flashpoint” of a liquid is the lowest temperature at which it can ignite in either liquid or vapor form. For example, a solvent with a flash point of 2 degrees Celsius is prone to ignition in almost any normal workplace, therefore needs to be handled with precautions, while a solvent with a flashpoint above 50 degrees Celsius would only be an ignition risk under specific extreme conditions. Flashpoints of some common solvents can be found in the following table.

Buffers

[0086] Methods for processing nucleic acids often involve exposing the nucleic acid to an aqueous solution, followed by precipitating the nucleic acid onto a solid support. Examples of such methods are provided in WO 2012/069660. An important component in such a method is the buffer used to precipitate the nucleic acid onto the solid support.

[0087] Provided herein are buffers that are useful in nucleic acid preparation, in particular for precipitation of nucleic acids from solution to a solid support, and/or for washing of a nucleic acid bound solid support.

[0088] An aspect provides an aqueous nucleic acid purification buffer comprising a polar aprotic solvent. These aqueous nucleic acid purification buffers provide a number of advantages when compared to solvents used in the prior art, such as alcohols and polyols. For example, the polar aprotic solvent is usually non-flammable and has low volatility, features that are not shared by common alcohols. This provides benefits such as improved safety for handling and shipping, e.g. allowing the buffer to be packaged and shipped in sealed containers and cartridges in ready to use format. The low volatility also provides for a reduced level of component concentration over time. The polar aprotic solvents typically have relatively low viscosity (e.g. viscosity of less than 50 cP at 20 °C and 1 atm), which allows for a good level of accuracy and ease when measuring and dispensing the buffer. The buffers may also provide other benefits, such as being relatively environmentally friendly (comprising components that are biodegradable and renewable), health and safety compliant, and being compatible with downstream nucleic acid processing/analysis procedures, such as amplification or mass spectrometry.

[0089] The polar aprotic solvent may be present in an amount of at least about 2 % wt. The polar aprotic solvent may be present in an amount of not more than about 80 % wt. For example, the polar aprotic solvent may be present in an amount of from between about 4 % wt to about 75 % wt.

[0090] The polar aprotic solvent may have a boiling point of more than about 100 °C at 1 atm. For example, the polar aprotic solvent may have a boiling point of more than about 150°C at 1 atm.

[0091] The polar aprotic solvent may comprise 2, 3 or 4 heteroatoms selected from O and N. For example, the polar aprotic solvent may comprise 2 or 3 heteroatoms selected from O and N. The polar aprotic solvent may comprise 2 or 3 O heteroatoms. The polar aprotic solvent may comprise 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. The polar aprotic solvent may comprise 2 O heteroatoms. The polar aprotic solvent may comprise 3 O heteroatoms. The polar aprotic solvent may comprise 1 O heteroatoms and 1 or 2 N heteroatoms. The O heteroatoms may comprise =0 and/or -O-.

[0092] The polar aprotic solvent may consist of carbon atoms (for example 4, 5, 6, 7, 8, 9 or 10 carbon atoms; e.g. 6, 7, 8 or 9 carbon atoms), hydrogen atoms and 2, 3 or 4 heteroatoms selected from O and N. For example, the polar aprotic solvent may comprise 2 or 3 heteroatoms selected from O and N. The polar aprotic solvent may comprise 2 or 3 O heteroatoms. The polar aprotic solvent may comprise 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. The polar aprotic solvent may comprise 2 O heteroatoms. The polar aprotic solvent may comprise 3 O heteroatoms. The polar aprotic solvent may comprise 1 O heteroatom and 1 or 2 N heteroatoms. The O heteroatoms may comprise =0 and/or -O-

[0093] The polar aprotic solvent may comprise 4, 5, 6, 7, 8, 9 or 10 carbon atoms. For example, the polar aprotic solvent may comprise 6, 7, 8, 9 or 10 carbon atoms; e.g. the polar aprotic solvent may comprise 6, 7 or 8 carbon atoms. For example, the polar aprotic solvent may comprise 5, 6, 7, or 8 carbon atoms; e.g. the polar aprotic solvent may comprise 6 or 8 carbon atoms. The polar aprotic solvent may comprise 5 carbon atoms. The polar aprotic solvent may comprise 6 carbon atoms. The polar aprotic solvent may comprise 7 carbon atoms. The polar aprotic solvent may comprise 8 carbon atoms.

[0094] The polar aprotic solvent may have a viscosity of less than about 50 cP at 20 °C and 1 atm. For example, the polar aprotic solvent may have a viscosity of less than about 40 cP or about 30 cP at 20 °C and 1 atm; e.g. the polar aprotic solvent may have a viscosity of less than about 25 cP at 20 °C and 1 atm. Viscosity may be measured in accordance with standard methods, for example according the method of OECD

(2012), Test No. 114: Viscosity of Liquids, OECD Guidelines for the Testing of Chemicals, Section 1, OECD Publishing, Paris, https://doi.Org/10.1787/9789264185180-en, the content of which is incorporated by reference herein. [0095] The polar aprotic solvent may have a flashpoint of at least about 50°C.

[0096] The polar aprotic solvent may be liquid at about 0 °C and 1 atm.

[0097] The polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N- Formylmorpholine. For example, the polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, and dipropylene glycol dimethyl ether.

[0098] The polar aprotic solvent may be or comprise N-butylpyrrolidin-2-one (Tamisolve™):

[0099] The polar aprotic solvent may be or comprise dihydrolevoglucosenone (Cyrene™):

[00100] The polar aprotic solvent may be or comprise dipropylene glycol dimethyl ether (Proglyde™):

[00101] The purification buffer may further comprise one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof. For example, the purification buffer may comprise two or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent, e.g. the purification buffer may comprise three or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent.

[00102] Exemplary chaotropes include guanidinium salts (such as GuSCN and GuHCI), urea, and the like. Surfactants may be selected from nonionic surfactants (such as Triton™ X-100, DDM, digitonin, Tween® 20, Tween® 80, Ecosurf™, and the like), anionic surfactants (such as sodium dodecyl sulfate, deoxycholate, cholate, sarkosyl, and the like), and zwitterionic surfactants (such as CHAPS, Zwittergent® 3-14, and the like). Buffering agents may be any suitable buffers that would provide buffering in the pH range of about 5 to about 9. Examples of suitable buffering agents include Tris, Trizma, Citrate, phosphate, Tricine, TAPS, PBS, acetate, borate, HEPES, Bicine, MOPS, CHES, carbonate, and the like, as well as mixtures thereof; and the buffer may also comprise other components such as polyethylene glycol (PEG), tetraethylene glycol (TEG), and linear polyacrylamide (LPA). Enzymes are typically protein or other degrading enzymes, such as proteinase K or lysozyme. Exemplary inorganic salts include metal halides, such as MgCh, NaCI, LiCI, and the like. However, in some embodiments the purification buffer may not contain a salt and/or a chaotrope.

[00103] The purification buffer may further comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of an alcohol, a polyol, a polyethylene glycol (PEG), tetraethylene glycol (TEG), or linear polyacrylamide (LPA), or a combination thereof. The purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of an alcohol or a polyol. The purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of a polyethylene glycol PEG, a TEG, or an LPA. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of alcohol, and/or polyol, and/or PEG, and/or TEG, and/or LPA may be preferred. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of alcohol, or polyol may be preferred.

[00104] The purification buffer may be a binding buffer and/or a wash buffer. For example, the purification buffer may be a binding buffer and a wash buffer. The purification buffer may be a binding buffer. The purification buffer may be a wash buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which only need to prevent (or at least minimize) the nucleic acid eluting from the solid support.

[00105] In view of this, the wash buffer may represent an aqueous dilution of the binding buffer. The aqueous dilution may also comprise other reagents that may not be present in the binding buffer, such as: a buffering agent, or a buffering agent and an inorganic salt. The wash buffer may comprise a 1.1 to 5 times dilution of the binding buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the binding buffer.

[00106] It follows that a workflow or kit could comprise both a binding buffer of the present invention and a wash buffer of the present invention. In some examples, the wash buffer may be the same as (or be an aqueous dilution of) the binding buffer. In other examples, the binding buffer and wash buffers have different compositions, i.e. the wash buffer is neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer. [00107] In embodiments, the purification buffer does not contain ethanol, isopropanol or 2- methyl-1,3- propanediol. In embodiments, the purification buffer does not contain dimethylsulfoxide (DMSO). In embodiments, the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or DMSO. In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol). In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol) or DMSO.

Methods and Uses

[00108] Figure 1A provides an exemplary schematic illustrating a general process for preparation of nucleic acids. In the first step a liquid sample is provided that comprises nucleic acid. Add a solid support and then a purification buffer (such as a binding buffer) to the liquid sample. Allow the nucleic acid to precipitation from solution to the solid support, to provide a nucleic acid bound solid phase. Separate the liquid from the bound solid support and wash the solid support one or more times with washing buffer(s), to provide a washed nucleic acid bound solid phase. Optionally dry the washed nucleic acid bound solid phase, to provide a dried nucleic acid bound solid phase. An elution buffer may be added to either the washed nucleic acid bound solid phase or the dried nucleic acid bound solid phase to provide an eluted nucleic acid. The eluted nucleic acid may be used in downstream nucleic acid processing I analysis. In addition, for some downstream application the elution step is not required, i.e. the washed nucleic acid bound solid phase or the dried nucleic acid bound solid phase applications may also be used in downstream nucleic acid processing I analysis.

[00109] Figure 1B illustrates a standard workflow that uses a purification buffer / binding buffer comprising isopropylalcohol (I PA), a Wash buffer 1 comprising 50% lysis/binding buffer (LBB) and 50% I PA, and a Wash buffer 2 comprising 70% ethanol. Figure 1C illustrates an exemplary workflow of the invention that uses a purification buffer I binding buffer comprising TamiSolve™, a Wash buffer 1 comprising 50% lysis/binding buffer (LBB) and 50% TamiSolve™, and a Wash buffer 2 comprising 50% TamiSolve™ or 50% 2- butoxyethanol (2-BE). The workflow of Figure 1C may also be performed with other exemplary polar aprotic solvents as disclosed herein (such as dihydrolevoglucosenone, dipropylene glycol dimethyl ether, and N-Formylmorpholine) instead of TamiSolve™.

[00110] The general process illustrated in Figure 1A may be preceded by preliminary extraction of the nucleic acid from a sample, for example by a lysis step as illustrated in Figures 1 B and 1C. Such lysis and binding of the nucleic acid thereby released to the solid support may employ the same lysis/ binding buffer, carried through from the lysis step to the binding step. [00111] The lysis or lysis/binding buffer typically comprises a chaotrope. Chaotropic agents denature macromolecules in the biological material, such as proteins and nucleic acids. Chaotropic agents also disrupt membrane lipids Thus, the chaotropic agent in the lysis solution functions to reduce enzymatic activity and facilitate the induction of cell lysis. Any suitable chaotropic agent may be used in the lysis or lysis/binding buffer. For instance, in some embodiments, the chaotropic agent is selected from a guanidinium salt (e.g. guanidinium isothiocyanate or guanidinium chloride), lithium perchlorate, lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea and a combination thereof. The chaotropic agent or chaotrope may be present in the lysis or lysis/binding buffer at a concentration sufficient to denature macromolecules in the biological material and/or induce cell lysis. In some embodiments, the lysis or lysis/binding buffer contains a guanidinium salt at a concentration of at least about 2M, at least about 3M, such as at least about 3.5M or about 4.0M, e.g about 3-6M. In a preferred embodiment, the lysis buffer contains guanidinium isothiocyanate at a concentration of about 3.5-4.5M, e.g. about 4.0M.

[00112] The lysis or lysis/binding buffer typically comprises a surfactant, for example a non-ionic surfactant or detergent, of which poly(oxyethylene)-containing surfactants may be mentioned. The detergent in the lysis solution functions to disrupt cellular and organelle membranes, e.g. lyse cells and organelles, and to denature proteins in the biological material. Thus, detergents function to facilitate the release of nucleic acids from cells and other entities, e.g. viruses, in the biological material. Any suitable detergent may be used in the lysis or lysis/binding solution, e.g. a non-ionic detergent. For instance, in some embodiments, the detergent is selected from sodium lauroyl sarcosinate (sarkosyl), sodium dodecyl sulfate (SOS) , 4-(1 , 1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, 2- [4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100) and a combination thereof. In a preferred embodiment, the detergent is selected from sodium lauroyl sarcosinate (sarkosyl) and 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100). The detergent is present in the lysis solution at a concentration sufficient to disrupt cellular and organelle membranes, e.g. lyse cells and organelles, disrupt viral envelopes and/or capsids, and/or to denature proteins in the biological material. In some embodiments, the detergent is present at a concentration of about 0.5-5.0% w/v, e.g. about 0.75-4.5% w/v, about 1.0-4.0% w/v, about 1.5-3.0% 20 w/v, such as about 1.75-2.25% w/v, e.g. about 2.0%. In some preferred embodiments, the detergent is sodium lauroyl sarcosinate (sarkosyl) or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol (Triton X-100) at a concentration as defined above.

[00113] The lysis/binding buffer may be any buffer known for this purpose, e.g. with a high concentration of chaotrope (for example a guanidinium salt or urea) and surfactant, e.g.

Triton x-100. For example, a preferred lysis buffer is a high detergent, guanidinium isothiocyanate (GTC)-containing buffer™.

[00114] In some instances, the lysis or lysis/binding buffer may comprise a citrate buffer, e.g. sodium or potassium citrate or may contain one or more chelating agent. Such chelating agents function to sequester divalent cations, which are essential for enzymes that act on nucleic acids, e.g. DNases and RNases. Thus, the chelating agent functions to inhibit or prevent the degradation of nucleic acids in the sample. Accordingly, the chelating agent comprises a chelator of divalent cations, for example EDTA (Ethylenediaminetetraacetic acid). The chelating agent may be present in the lysis solution at a concentration sufficient to inhibit nucleic acid degrading enzymes (i.e. in the sample comprising biological material), e.g. about 5-50 mM, such as about 10-40 mM or about 15- 30 mM, e.g. about 20 mM. Thus, in some embodiments, the lysis solution comprises EDTA at a concentration as defined above.

[00115] A lysis or lysis/binding buffer may also contain one or more reducing agent. When present in the lysis buffer, the reducing agent functions to reduce disulfide bonds in proteins in the biological material. Suitable reducing agents are well-known in the art and may be selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (OTT), p- mercaptoethanol (P-ME) and a combination thereof. The use of TCEP may be particularly advantageous because it has high stability and activity at room temperature, thereby facilitating the production of a lysis buffer with improved activity that can be stored long term (e.g. useful for large scale production of a commercial product). Thus, in some preferred embodiments, the reducing agent is TCEP.

[00116] The reducing agent, when included in the lysis or lysis/binding buffer, may be present at a concentration sufficient to reduce disulfide bonds in proteins in the biological material. In some embodiments, the reducing agent may be present at a concentration of about 1-20mM, such as about 2-19mM, 3-18mM, 4-17mM or about 5-16mM, e.g. about 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15mM, preferably about 10 10mM. However, in some embodiments, a higher amount of reducing agent may be used, e.g. about 20-150mM, such as about 25-125mM or about 30-1 OOmM, e.g about 80mM.

[00117] Furthermore, a lysis or lysis/binding buffer suitable in methods and uses of the invention may comprise a “nucleic acid carrier” which functions to increase the concentration of nucleic acids in the sample. In some embodiments this may facilitate aggregation of nucleic acids by the precipitating agent and adsorption onto the solid support (e.g. silica-coated magnetic beads). Nucleic acid carriers typically are polymers, such as nucleic acids or polysaccharides. For instance, a nucleic acid carrier may be selected from glycogen, sonicated DNA (e.g. sonicated calf thymus or salmon sperm DNA), poly dT and/or poly dA, tRNA, polyacrylamide (e.g. linear polyacrylamide) and a combination thereof. In some examples, the use of glycogen may be particularly advantageous because it is an inert molecule that does not interfere with downstream nucleic acid reactions, e.g. amplification and/or detection reactions. Thus, in some preferred embodiments, the nucleic acid carrier may be glycogen.

[00118] The nucleic acid carrier, when included in the lysis or lysis/binding buffer, may be present at a concentration sufficient to increase the recovery of nucleic acids from the biological material, i.e. to increase the adsorption of the nucleic acids on a solid support. In some embodiments, the nucleic acid carrier may be present in the lysis buffer at a concentration of about 0.1-5 mg/ml, such as about 0.2-4.0 mg/ml, 0.3-4.0 mg/ml, 0.4- 3.0 mg/ml or about 0.5-3.0 mg/ml, e.g. about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4 or 1.5 mg/ml, preferably about 1.0mg/ml.

[00119] The lysis step may also use other components such as a protease, e.g. proteinase K. In some instances, a proteinase may enhance the efficiency of nucleic acid extraction. The lysis may therefore be performed by combining a cell, a virus or another biological structure with a chaotrope (for example a guanidinium salt or urea), a surfactant (for example a non-ionic surfactant, e.g. a polyoxyethylenic surfactant) and a protease (such as a proteinase, for example proteinase K). In some embodiments, a protease (such as a proteinase) may be included in the lysis buffer. Alternatively, in some embodiments, a protease (such as a proteinase) may be added to the sample after it has been contacted with the lysis buffer but before it is contacted with the binding or purification buffer. In some embodiments, a protease (such as a proteinase) may be added contemporaneously when contacting the sample with a solid support (e.g. with a suspension of magnetic particles).

[00120] In the nucleic acid-binding step of a conventional nucleic acid isolation protocol, the lysis/ binding buffer is commonly supplemented with isopropanol e.g. 50% isopropanol, or it may be supplemented with ethanol, to facilitate capture of the nucleic acid on the solid support. This, however, is not ideal, for example because isopropanol is volatile and flammable.

[00121] An aspect of the invention provides the use of a purification buffer of the first aspect, to precipitate a nucleic acid from solution to a solid support, thereby providing a nucleic acid bound solid support.

[00122] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties. The surface hydroxyl functional groups may comprise part of silanol moieties. The surface hydroxyl functional groups may comprise part of carboxyl moieties. The surface hydroxyl functional groups may comprise part of saccharide moieties. The saccharide moieties may be or comprise one or more polysaccharides, e.g. a complex branched glucan such as dextran.

[00123] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The beads may be monodisperse and I or magnetic and I or porous. The (optionally monodisperse) beads may be magnetic and I or porous. The beads may be monodisperse; as well as magnetic and I or porous. The beads may be magnetic, e.g. the beads may be monodisperse and magnetic.

[00124] In some instances, the magnetic beads may comprise microparticles or nanoparticles. In some examples, the magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application No. GB2210796.5 which is hereby incorporated by reference may be used. In some examples the solid support may be selected from any commercially available solid support suitable for binding nucleic acid. For example, the solid support may be selected from Dynabeads™ MyOne™ Silane, Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M-270™ Carboxylic Acid, Dynabeads™, Oligo(dT)25 magnetic beads (all available from Thermo Fisher Scientific), SeraMag SpeedBeads™ carboxylate-modified or SeraSil-Mag 400 or 700 (Cytiva), BioMagPlus COOH™ and ProMag 1 COOH™ (both Bangs Laboratories, INC Fishers), 4.4 pm fluorescent ferromagnetic beads or 2.0 pm ferromagnetic beads (both available from Spherotech INC Lake Forest, IL), 2 pm beads designated WHM-S001™ or 2 pm beads designated WHM-S002™ (both available from Creative Diagnostics, New York, NY), Silicon Hydroxyl Magnetic Microspheres or Carboxyl Magnetic Microspheres or Oligo(dT) Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), Carboxyl Adembeads (available at 100 nm, 200 nm, 300 nm or 500 nm) or Carboxyl Masterbeads (500 nm) (available from Ademtech, France), MagneSil™ beads (available from Promega), BeaverBeads™ Mag COOH (available from Beaver Biomedical Engineering Ltd.), Lodestars High Bind Carboxyl beads (available from Agilent), MagnosphereTM, MS300 Carboxyl, MS 160 Carboxyl or MS160 Carboxyl (all available from JSR Life Sciences), PureProteome Carboxy FlexiBind Magnetic Bead System (available with different bead sizes from Sigma-Aldrich), BioMag™ Carboxl, BioMag™ Maxi Carboxyl or BioMag™ Plus Carboxyl (all available from Polysciences), Carboxyl Super Mag or Mono Mag Magnetic Beads (available at sizes between 0.1 pm and 4.5 p from Ocean Nanotech), and Carboxyl beads of different sizes available from VdoBiotech. [00125] The use may further comprise washing the nucleic acid bound solid support with a wash buffer. The wash buffer may be or comprise the purification buffer; optionally in admixture with other components. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding. For example, the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.

[00126] The use may further comprise contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support. The elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof. A conventional elution buffer may be used, for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 Mm Tris-HCI buffer at pH 8.0.

[00127] The separated nucleic acid may be further subjected to one or more additional processes. Such a separated nucleic acid may be subjected to said additional processes when it is bound to the solid support (optionally after washing the nucleic acid bound solid support), or after it has been separated from the solid support.

[00128] The one or more additional processes may be selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

[00129] The amplification may comprise PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. The isothermal amplification may comprise loop- mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicasedependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA) (for an overview see “Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", Infection and Drug Resistance 2020:13, 455-483 and references therein).

[00130] The sequencing may comprise next generation sequencing. “Next-generation sequencing” and “high-throughput sequencing” are sequencing techniques that parallelize the sequencing process, producing a high number of sequences at once. The nextgeneration sequencing methods may comprise single molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 Life Sciences), sequencing by ligation (e.g., SOLiD sequencing of Life Technologies), sequencing by synthesis and reversible terminator (e.g., Illumina), reversible dye terminator-based sequencing (e.g. Solexa), Oxford Nanopore sequencing, FRET-donor-polymerase-based sequencing (VisiGen Biotechnologies), extension-based single molecule sequencing (Helicos Bioscience), hybridization sequencing, nucleic acid imaging technologies such as transmission electron microscopy, and the like.

[00131] The nucleic acid may be DNA. The DNA may, for example, be one or more of synthetic DNA (e.g. DNA assembled from synthetic oligonucleotides, enzymatically synthesized DNA, doggy-bone, dumb-bell DNA etc.), plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA.

[00132] The nucleic acid may be RNA. The RNA may, for example, be one or more of mRNA (e.g. isolated from a biological sample or in v/fro-transcribed), siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).

[00133] Another aspect provides a method for processing a nucleic acid. The method comprises exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support.

[00134] In preferred embodiments, the aqueous medium comprising the polar aprotic solvent is an aqueous nucleic acid purification buffer as defined in the first aspect.

[00135] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties. The surface hydroxyl functional groups may comprise part of silanol moieties. The surface hydroxyl functional groups may comprise part of carboxyl moieties. The surface hydroxyl functional groups may comprise part of saccharide moieties. The saccharide moieties may be or comprise one or more polysaccharides, e.g. a complex branched glucan such as dextran.

[00136] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The beads may be monodisperse and I or magnetic and I or porous. The (optionally monodisperse) beads may be magnetic and I or porous. The beads may be monodisperse; as well as magnetic and I or porous. The beads may be magnetic, e.g. the beads may be monodisperse and magnetic. The (optionally monodisperse) beads may be magnetic.

[00137] The method may further comprise washing the nucleic acid bound solid support with a wash buffer. The wash buffer may be or comprise the purification buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding. For example, the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.

[00138] The method may further comprise contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid. The elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof. A conventional elution buffer may be used, for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 Mm Tris-HCI buffer at pH 8.0.

[00139] The method may further comprise subjecting the nucleic acid (e.g. the separated nucleic acid) to one or more additional processes. Such a separated nucleic acid may be subjected to said additional processes when it is bound to the solid support (optionally after washing the nucleic acid bound solid support), or after it has been separated from the solid support.

[00140] The one or more additional processes may be selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation, and genome editing.

[00141] The amplification may comprise PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. The isothermal amplification may comprise loop- mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicasedependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA) (for an overview see “Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", Infection and Drug Resistance 2020:13, 455-483 and references therein).

[00142] The sequencing may comprise next generation sequencing. “Next-generation sequencing” and “high-throughput sequencing” are sequencing techniques that parallelize the sequencing process, producing a high number of sequences at once. The nextgeneration sequencing methods may comprise single molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 Life Sciences), sequencing by ligation (e.g., SOLiD sequencing of Life Technologies), sequencing by synthesis and reversible terminator (e.g., Illumina), reversible dye terminator-based sequencing (e.g. Solexa), Oxford Nanopore sequencing, FRET-donor-polymerase-based sequencing (VisiGen Biotechnologies), extension-based single molecule sequencing (Helicos Bioscience), hybridization sequencing, nucleic acid imaging technologies such as transmission electron microscopy, and the like.

[00143] The sample may comprise or be a pre-treated or untreated biological sample, environmental sample, or an enzymatic reaction mixture. The sample may be or comprise a pre-treated or untreated biological sample. The biological sample may be in a physiological buffer or transport medium. The sample may be or comprise an environmental sample. The sample may be or comprise an enzymatic reaction mixture.

[00144] The biological sample may be any suitable biological sample. Exemplary biological samples may comprise one or more of blood, blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin-fixed paraffin- embedded (FFPE) sample, biopsy sample, bone marrow or other tissue sample, plant sample, cell lysate, bacterial or yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk. In some instances, the biological sample may be a clinical sample. In some instances, the sample may be a cell-free sample.

[00145] The environmental sample may comprise a water sample (such as a wastewater sample), a soil sample, a sediment sample, a surface swab, an air derived sample (such as an air filter residue), a cosmetic, a food ingredient or a food sample, or a combination thereof.

[00146] The enzymatic reaction mixture could be any such mixture that may contain nucleic acid sequences. For example, the enzymatic reaction mixture may comprise in vitro transcription reaction mixture, a reverse transcription reaction mixture, a second strand synthesis reaction mixture, an amplification reaction mixture, a library preparation reaction mixture, a restriction reaction mixture, a nucleic acid assembly reaction mixture, or a barcoding reaction mixture.

[00147] In embodiments, the aqueous medium does not contain ethanol, isopropanol or 2- methyl-1 ,3- propanediol. In embodiments, the aqueous medium does not contain dimethylsulfoxide (DMSO). In embodiments, the aqueous medium does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or DMSO. In embodiments, the aqueous medium does not contain an alcohol (including any diol or polyol). In embodiments, the aqueous medium does not contain an alcohol (including any diol or polyol) or DMSO.

[00148] The method may further comprise performing point-of-care (POC) detection. The point-of-care detection may comprise pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection, and/or can be utilized in genetic screening. The pathogen detection or biological warfare agent detection may comprise viral, bacterial, single-celled fungi or protozoan nucleic acid detection.

[00149] Point-of-care detection may be performed using a point-of-care instrument. The point-of-care detection may comprise use of a cartridge or microfluidic chip. The cartridge may comprise compartments containing one or more of the solid support, aqueous medium comprising a polar aprotic solvent (e.g. aqueous nucleic acid purification buffer as disclosed herein), wash buffer, and elution buffer. The cartridge may also comprise the necessary microfluidics to transfer reagents between compartments to perform the methods of processing nucleic acids described herein. Such cartridges may be typically shipped and stored in ready to use format, so it is advantageous if reagents (such as aqueous medium comprising a polar aprotic solvent and/or buffers) contained therein are not flammable.

[00150] An exemplary point-of-care-detection method may comprise introducing the sample to a cartridge or microfluidic chip, performing the following steps on the cartridge: exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support; washing the nucleic acid bound solid support with a wash buffer; and contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid; and then subjecting the nucleic acid to one or more additional processes. Examples of additional processes that may be relevant to point-of -care detection include detection, cloning, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography, and mass spectrometry.

[00151] Examples of point-of-care instruments that may be adapted for use in accordance with the methods of the present disclosure are provided in US 9,752,182 B2 and US 2016/0016171 A1 , the content of which is incorporated by reference herein.

Kits, Compositions and Apparatus

[00152] An important advantage of the present invention is that the polar aprotic solvents used in the present invention can be used in buffers that are non-hazardous and therefore well suited to be shipped in kits.

[00153] Accordingly, an aspect of the invention is a kit comprising an aqueous nucleic acid purification buffer of the present invention (e.g. of the first aspect), and a solid support.

[00154] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties. The surface hydroxyl functional groups may comprise part of silanol moieties. The surface hydroxyl functional groups may comprise part of carboxyl moieties. The surface hydroxyl functional groups may comprise part of saccharide moieties. The saccharide moieties may be or comprise one or more polysaccharides, e.g. a complex branched glucan such as dextran.

[00155] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. The solid support may comprise beads, for example monodisperse beads. The beads may be monodisperse and I or magnetic and I or porous. The (optionally monodisperse) beads may be magnetic and I or porous. The beads may be monodisperse; as well as magnetic and I or porous. The beads may be magnetic, e.g. the beads may be monodisperse and magnetic.

[00156] The kit may comprise one or more of a lysis or lysis/binding buffer, a wash buffer, and an elution buffer. The lysis or lysis/binding buffer and/or the wash buffer may comprise the polar aprotic solvent of the aqueous nucleic acid purification buffer.

[00157] The lysis or lysis/binding buffer may comprise a high concentration of chaotrope (for example a guanidinium salt or urea) and surfactant, e.g. Triton x-100. The lysis or lysis/binding buffer may comprise any of the components disclosed in connection with methods, uses and compositions herein. The lysis buffer may comprise enzymes, e.g. lysozyme, proteolytic enzymes (such as or proteinase K) and the like.

[00158] The wash buffer may be or comprise the purification buffer. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding. The aqueous dilution may also comprise other reagents that may not be present in the purification buffer that is used for binding, such as: a buffering agent, or a buffering agent and an inorganic salt. The wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer; e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.

[00159] In some examples, the wash buffer may be the same as (or be an aqueous dilution of) the purification buffer. In other examples, the purification buffer and wash buffers have different compositions, i.e. the wash buffer is nether the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.

[00160] The elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof. A conventional elution buffer may be used, for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 Mm Tris-HCI buffer at pH 8.0.

[00161] Another aspect provides a composition of matter. The composition of matter comprises (i) a solution comprising an aqueous nucleic acid purification buffer of the invention (e.g. in accordance with the first aspect); and (ii) a nucleic acid solid support.

[00162] The solid support may comprise surface hydroxyl functional groups. The surface hydroxyl functional groups may comprise part of silanol, carboxyl, or saccharide moieties. The surface hydroxyl functional groups may comprise part of silanol moieties. The surface hydroxyl functional groups may comprise part of carboxyl moieties. The surface hydroxyl functional groups may comprise part of saccharide moieties. The saccharide moieties may be or comprise one or more polysaccharides, e.g. a complex branched glucan such as dextran.

[00163] The solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc or a slide. The solid support may comprise beads, for example monodisperse beads. The beads may be monodisperse and I or magnetic and I or porous. The (optionally monodisperse) beads may be magnetic and I or porous. The beads may be monodisperse; as well as magnetic and I or porous. The beads may be magnetic, e.g. the beads may be monodisperse and magnetic.

[00164] The solution (i) may further comprise lysis or lysis/binding buffer related components, such as one or more of the following: a chaotrope (for example a guanidinium salt or urea); a surfactant, e.g. Triton x-100; and one or more enzymes, for example lysozyme, a proteolytic enzyme (such as proteinase K) and the like. The solution (i) may comprise any of the components disclosed in connection with methods, uses and kits herein. [00165] The composition of matter may further comprise a sample, such as a pre-treated or untreated biological sample, environmental sample, or an enzymatic reaction mixture, as described herein; said sample comprising a nucleic acid.

[00166] The nucleic acid may be DNA; such as plasmid DNA, cDNA, cfDNA and I or genomic DNA. The nucleic acid may be RNA; such as one or more of mRNA (e.g. isolated from a biological sample or in v/tro-transcribed), siRNA, microRNA, tRNA, or cfRNA.

[00167] A further aspect provides use of a kit of the invention in an automated nucleic acid analysis platform. Automated nucleic acid analysis system has a benefit of analysing large number of samples in parallel during reduced period of time, eliminates the risk of manual processing errors during the analysis and requires minimal operator hands-on time. Kits of the invention may be used in both automated liquid handling, pipetting systems or in automated multi-purpose, high-throughput integrated laboratory systems that include downstream processing functionalities. Exemplary automated nucleic acid analysis platforms to be used with the present invention could be including, but not limited to, liquid handling and automation systems such as DreamPrep™ NAP workstation (TECAN), Fluent™ Automation Workstation (TECAN), Microlab Prep, NIMBUS, STAR or VANTAGE pipetting platforms (Hamilton Company). Additionally, compatible automated systems could provide downstream processing, such as genotyping or diagnostics testing, e.g. COR™ MX/PX or GX System (Beckton Dickinson), COBAS™ 5800 system (Roche Diagnostics)

[00168] A seventh aspect provides a nucleic acid analysis apparatus. The apparatus comprises an automated nucleic acid analysis platform and a kit of the fourth aspect. The automated nucleic acid analysis platform comprises a portion configured to house the solid support.

[00169] The automated nucleic acid analysis platform may be a point-of care assay instrument. The point-of-care assay instrument may be adapted for pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection. The pathogen detection or biological warfare agent detection may comprise viral or bacterial nucleic acid detection. Exemplary point-of care assay instruments include ePlex® systems available from GenMark Diagnostics, Inc. of Carlsbad, CA, USA, and Solana® instruments available from Quidel of San Diego, CA, USA. Further examples of point-of-care assay instruments are provided in US 9,752,182 B2 and US 2016/0016171 A1 , the content of which is incorporated by reference herein.

Additional Embodiments

[00170] The invention and disclosure also comprises the subject matter of the following clauses:

1. An aqueous nucleic acid purification buffer comprising a polar aprotic solvent.

2. The purification buffer of clause 1 , wherein the polar aprotic solvent is present in an amount of at least about 2 %wt.

3. The purification buffer of clause 1 or clause 2, wherein the polar aprotic solvent is present in an amount of not more than about 80 %wt.

4. The purification buffer of any preceding clause, wherein the polar aprotic solvent is present in an amount of from between about 4 %wt to about 75 %wt.

5. The purification buffer of any preceding clause, wherein the polar aprotic solvent has a boiling point of more than 100 °C at 1 atm, optionally a boiling point of more than 150°C at 1 atm.

6. The purification buffer of any preceding clause, wherein the polar aprotic solvent comprises 2, 3 or 4 heteroatoms selected from O and N; optionally wherein the polar aprotic solvent comprises 2 or 3 heteroatoms selected from O and N; and/or optionally wherein the polar aprotic solvent consists of carbon atoms, hydrogen atoms and 2, 3 or 4 heteroatoms selected from O and N.

7. The purification buffer of any preceding clause, wherein the polar aprotic solvent comprises 4, 5, 6, 7, 8, 9 or 10 carbon atoms; optionally wherein the polar aprotic solvent comprises 5, 6, 7, or 8 carbon atoms; or optionally wherein the polar aprotic solvent comprises 6, 7, 8, 9, or 10 (e.g. 6, 7, or

8) carbon atoms.

8. The purification buffer of any proceeding clause, wherein the polar aprotic solvent has a viscosity of less than 50 cP at 20 °C and 1 atm; optionally wherein the polar aprotic solvent has a viscosity of less than 25 cP at 20 °C and 1 atm.

9. The purification buffer of any proceeding clause, wherein the polar aprotic solvent has a flashpoint of at least 50 °C.

10. The purification buffer of any proceeding clause, wherein the polar aprotic solvent is liquid at 0 °C and 1 atm.

11. The purification buffer of any preceding clause, wherein the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine. 12. The purification buffer of any preceding clause, wherein the purification buffer further comprises one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof.

13. The purification buffer of any preceding clause, wherein the purification buffer is a binding buffer and/or a wash buffer.

14. The wash buffer of clause 13, wherein the wash buffer comprises a 1.1 to 5 times dilution of the binding buffer.

15. The use of a purification buffer of any of clauses 1 to 12 to precipitate a nucleic acid from solution to a solid support, thereby providing a nucleic acid bound solid support.

16. The use of clause 15, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties.

17. The use of clause 15 or 16, wherein the solid support comprises one or more of particles, resin, cartridge, beads, a filter, a column, an array, a membrane, a chip, a disc, or a slide.

18. The use of any of clauses 15 to 17, wherein the solid support comprises beads, optionally monodisperse beads.

19. The use of clause 18, wherein the (optionally monodisperse) beads are magnetic.

20. The use of any of clauses 15 to 19, further comprising washing the nucleic acid bound solid support with a wash buffer.

21. The use of clause 20, wherein the wash buffer is or comprises the purification buffer.

22. The use of any of clauses 15 to 21, further comprising contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support.

23. The use of clause 22, wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.

24. The use of clauses 22-23, wherein the separated nucleic acid is further subjected to one or more additional processes, optionally selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing. The use of clause 24, wherein the amplification comprises PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. The use of clause 25, wherein the isothermal amplification comprises loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA). The use of any of clauses 15 to 24, wherein the nucleic acid is DNA; optionally one or more of synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA. The use of any of clauses 15 to 24, wherein the nucleic acid is RNA; optionally one or more of mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA). A method for processing a nucleic acid, the method comprising: exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support. The method of clause 29, wherein the aqueous medium comprising the polar aprotic solvent is an aqueous nucleic acid purification buffer as defined in any of clauses 1 to 14. The method of clause 29 or clause 30, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties. The method of any of clauses 29 to 31 , wherein the solid support comprises one or more of particles, resin, cartridge, beads, a filter, a column, an array, a membrane, a chip, a disc, or a slide; optionally wherein the solid support comprises (optionally monodisperse) beads. The method of any of clauses 29 to 32, further comprising washing the nucleic acid bound solid support with a wash buffer; optionally wherein the wash buffer is or comprises the purification buffer. 34. The method of any of clauses 29 to 33, further comprising contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid.

35. The method of any of clauses 29 to 34, wherein the nucleic acid is further subjected to one or more additional processes, optionally selected from detection, cloning, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridisation, cDNA synthesis, size separation, chromatography, mass spectrometry, pharmaceutical or therapeutic formulation, and genome editing.

36. The method of any of clauses 29 to 35, wherein the sample comprises or is a pretreated or untreated biological sample, environmental sample, or an enzymatic reaction mixture.

37. The method of clause 36, wherein the environmental sample comprises water sample, optionally wastewater sample, soil sample, sediment sample, surface swab, an air derived sample (such as an air filter residue), a cosmetic, a food ingredient or a food sample or a combination thereof.

38. The method of clause 36, wherein the enzymatic reaction mixture comprises in vitro transcription reaction mixture, reverse transcription, second strand synthesis reaction mixture, amplification reaction mixture, library preparation reaction mixture, restriction reaction mixture, nucleic acid assembly reaction mixture, barcoding reaction mixture.

39. The method of clause 36, wherein the biological sample comprises one or more of blood, blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin-fixed paraffin-embedded (FFPE) sample, biopsy sample, bone marrow, tissue sample, plant sample, cell lysate, bacterial or yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, exhalates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk, optionally in a physiological buffer or transport medium.

40. The method of any of clauses 29-39, wherein the aqueous medium does not contain ethanol, isopropanol or 2-methyl-1 ,3- propanediol.

41 . The method of any of clauses 29-40, further comprising performing point-of-care detection. 42. The method of clause 41 , wherein the point-of-care detection comprises pathogen detection, biological warfare agent detection or genetic disease detection.

43. The method of clause 42, wherein pathogen detection or biological warfare agent detection comprise viral or bacterial nucleic acid detection.

44. A kit comprising an aqueous nucleic acid purification buffer as defined in any of clause 1 to 14, and a solid support.

45. The kit of clause 44, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties; and I or wherein the solid support comprises one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane or a chip, a disc, or a slide; optionally wherein the solid support comprises (optionally monodisperse) beads.

46. The kit of clause 44 or clause 45, further comprising one or more of a lysis buffer, a wash buffer, and an elution buffer; optionally wherein the lysis buffer and/or the wash buffer comprise the polar aprotic solvent of the aqueous nucleic acid purification buffer.

47. A composition of matter comprising:

(i) a solution comprising an aqueous nucleic acid purification buffer as defined in any of clauses 1 to 14; and

(ii) a nucleic acid solid support.

48. The composition of clause 47, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties; and I or wherein the solid support comprises one or more of particles, resin, beads, a filter, a column, a cartridge, an array, a membrane, a chip, a disc, or a slide; optionally wherein the solid support comprises (optionally) monodisperse beads.

49. The composition of clause 47 or clause 48, wherein the nucleic acid is DNA; optionally plasmid DNA, cDNA, cfDNA and I or genomic DNA; or wherein the nucleic acid is RNA; optionally one or more of mRNA, siRNA, microRNA, tRNA, or cfRNA.

50. Use of a kit of any of clauses 44 to 46 in an automated nucleic acid analysis platform.

51. A nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of clauses 44 to 46, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.

52. The nucleic acid analysis apparatus of clause 51 , wherein the automated nucleic acid analysis platform is a point-of care assay instrument.

53. The nucleic acid analysis apparatus of clause 52, wherein the point-of-care assay instrument is adapted for pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection.

Examples

Example 1 : Protocol for Viral Nucleic Acid (vNA) preparation with silane beads

[00171] Reagents:

• Silane magnetic beads Dynabeads™ MyOne SILANE (Thermo Fisher Scientific).

• Aqueous NA purification buffer comprising guanidine salt and Tween.

• Trigger: aprotic solvent for nucleic acid purification buffer, as set out in Table 1 for embodiments. Isopropanol (I PA) was used as the alternative trigger for positive controls.

• Wash buffer 1 : 50% Silane Rare Target NA Purification Buffer and 50% I PA. Note that similar results would be expected if the 50% I PA was replaced with about 50% of aprotic solvent used in trigger.

• Wash buffer 2: 70% ethanol in water. Note that similar results would be expected if the 70% ethanol was replaced with about 70% trigger.

• Elution buffer: Tris pH 8.

• LB Buffer comprising guanidine salt, ionic or non-ionic detergent (e.g. a Tween, such as Tween 20), pH 5-9.

• Proteinase K, at 20 mg/mL.

[00172] T able 1 : T rigger parameters for T rigger testing: 150 pL of T rigger was added at below dilutions at step 6 in the protocol

[00173] Consumables and equipment:

• Magnetic separator: DynaMag™- 2 magnet, SKU 12321 D (Thermo Fisher Scientific). This device provides a rack for microtubes, with a magnet in its base to provide efficient separation of magnetic beads from supernatant.

• 1.5 mL microtubes.

• Tube roller/rotator.

• Microtube heating block (pre-heated to desired lysis and/or elution temperature).

• Microtube bench top centrifuge.

• 1000 pL, 200 pL, and 100 pL pipettors.

• Timer.

• Thermal mixer/shaker.

• Optional equipment, for highly efficient manual processing: o Laboratory vacuum pump for supernatant removal; o Single use glass Pasteur pipettes with long capillary tip.

[00174] Procedure:

1. Allow buffers to reach room temperature prior to use. Remove silane magnetic beads from refrigerator. Vortex bottle for 20 seconds in order to re-suspend beads. Rotate bottle on a roller/rotator at room temperature for 20 minutes prior to use in Step 6.

2. Add 200 pL serum/plasma/UTM (Universal Transport Medium) sample to a 1.5 mL tube.

3. Add 50 pL Proteinase K. Mix with a brief pulse-vortex.

4. Add 300 pL LB Buffer. Mix with a brief pulse-vortex and then incubate for 10 minutes at room temperature. For thermal mixer/shaker, mix 10 min. 900 rpm.

5. Add 25 pL Dynabeads MyOne SILANE (1 mg) or Dynabeads MyOne Carboxylic Acid (1 mg). Mix with a brief pulse-vortex.

6. Gently add 150 pL Triggerto the lysate. Mix with a brief pulse-vortex. Incubate the tube on a rotator/roller at room temperature for 10 minutes. For thermal mixer/shaker, mix 10 min. 900 rpm.

7. Place the tube onto the DynaMag™-2 magnet. Allow beads to collect to magnet for 1-2 minutes or until completely cleared.

8. Using a 1000 pL pipettor or a vacuum pump, carefully remove the lysate and discard. Remove tube from magnet.

9. Wash Buffer 1 first wash: Add 800pl Wash Buffer 1 and vortex until the beads are fully resuspended. Place the tube onto the DynaMag™-2 magnet. Allow beads to collect to magnet for 1-2 minutes.

10. Using a 1000 pL pipettor or a vacuum pump, carefully remove Wash Buffer 1 and discard.

11. Wash Buffer 1 second wash: Perform a second wash by repeating Steps 9-10. Note: Use the same volume of Wash Buffer 1 as in Step 9.

12. Wash Buffer 2 first wash: Remove tube from magnet and then add 500 pL Wash Buffer 2. Mix by vortex/pi petti ng until the beads are fully resuspended. Transfer the bead solution using a 1000 pL pipettor to a fresh 1.5 mL microtube placed on the DynaMag™-2 magnet.

13. Allow beads to collect to magnet for 1 minute. Carefully remove Wash Buffer 2 using a pipettor with a 1000 pL tip or a vacuum pump from the 1.5 mL tube and discard.

14. Wash Buffer 2 second wash: Add 500 pL Wash Buffer 2.

15. Remove tube from magnet. Mix by vortex/shake until the beads are fully resuspended and then collect bead solution by brief centrifugation (1 second pulse). Take care not to pellet beads.

16. Place tube back onto the DynaMag™-2 magnet. Allow beads to collect to magnet for 30 seconds or until completely cleared. Carefully remove supernatant using a pipettor with a 1000 pL pipettor or a vacuum pump and discard.

17. To remove the trapped fluid between the beads, tap down the rack/magnet with the tube onto the bench top 3-5 times with medium force. Use a small pipette to aspirate the final droplets and discard. All fluid should be removed prior to elution.

18. Add 100 pL Elution Buffer.

19. Vortex between 20 seconds and 1 minute until the bead pellet is fully resuspended. Collect bead solution using brief centrifugation (1 second pulse). Take care not to pellet beads.

20. Incubate in a pre-heated block at 80 °C for 5 minutes. For thermal mixer/shaker, at 80 °C mix 5 min. 1500 rpm.

21. Vortex for 15 seconds, then collect bead solution by brief centrifugation (1 second pulse).

22. Place tube on the DynaMag™-2 magnet. Allow beads to collect to magnet for 15 seconds.

23. Transfer the eluted nucleic acid to a fresh 1.5 mL tube. Store at the appropriate temperature (e.g. DNA: 4 °C to -80 °C).

Example 2: Protocol for Viral Nucleic Acid (vNA) preparation with carboxylic acid beads

[00175] The protocol of Example 2 is as per the protocol of Example 1, but with the “Silane magnetic beads” replaced with the following reagent:

• Carboxylic acid magnetic beads: Dynabeads™ MyOne™ Carboxylic Acid (Thermo Fisher Scientific).

Example 3: Assessment of purification buffer comprising polar aprotic solvent with silane beads and carboxylic acid beads

[00176] The efficiency of exemplary purification buffers comprising a polar aprotic solvent for nucleic acid purification was assessed. In order to study the efficiency of these buffers in triggering nucleic acid binding to either a silanol or carboxylic acid functionalized surfaces, M13 bacteriophage (1.4 e5 pfu) and MS2 RNA (1.5 e6 copies) at 1.4 e5 pfu and 1 ,5e6 copies, respectively were spiked into 200pL human plasma prior to sample processing, and then processed according to the protocols of Example 1 and Example 2. The eluted samples were then analyzed by qPCR to quantify the M 13 DNA recovery or RT-qPCR to quantify the MS2 RNA recovery.

[00177] Figure 3 provides results that illustrate the recovery of nucleic acids from silanol functionalised stationary phases using aprotic solvents selected from Gyrene™, Proglyde™, TamiSolve™, or N-formylmorpholine (N-FM) in the binding buffer, with the protic solvent isopropanol (I PA) used as a positive control. (A) indicates the MS2 RNA recovery on silane beads, while (B) indicates the M 13 DNA recovery on silane beads.

[00178] Figure 4 provides results that illustrate the efficiency of binding to and recovery of nucleic acids from carboxylic acid functionalised stationary phases using aprotic solvents selected from Gyrene™, Proglyde™, TamiSolve™, or N-Formylmorpholine (N-FM) in the binding buffer, with the protic solvent isopropanol (I PA) used as a positive control. (A) indicate the MS2 RNA recovery on carboxy beads, while (B) indicates the M 13 DNA recovery on carboxy beads. [00179] As the results in Figures 3 and 4 show, all of the purification buffers comprising polar aprotic solvent cause NA to precipitate I bind to silane or carboxylic acid beads, albeit to different degree. The data show that Proglyde™ and TamiSolve™ show promise as aprotic solvents for use in nucleic acid binding buffer or wash buffer for both silane and carboxylic acid surfaces, although more efficiently with carboxylic acid surfaces in tested buffer compositions. Gyrene™ is the most efficient on silane beads (data for M 13 for Gyrene™ on silane beads not shown). N-Formylmorpholine worked well with DNA target on silane and less efficiently for RNA targets on silane, indicating that it may be useful for selective purification of DNA in a sample containing both DNA and RNA.

Example 4: Fully aprotic solvent based workflow

[00180] The efficiency of exemplary purification workflow wherein all buffers comprise a polar aprotic solvent for nucleic acid purification was assessed.

[00181] In order to study the efficiency of these buffers in triggering nucleic acid binding to and washing the nucleic acid molecules bound to or carboxylic acid functionalized surfaces, approximately 100 copies of a 120 bp DNA model and MS2 RNA, respectively were spiked into 200pL human plasma and then processed according to the protocols of Example 1 and Example 2.

[00182] Isopropanol and/or ethanol were replaced by TamiSolve™ and/or 2-butoxyethanol as given in Table 2 below. The resulting eluate was analyzed by qPCR or RT-qPCR.

[00183] Table 2: Solvents and buffers used in fully aprotic workflow and other extractions

[00184] Figures 5a and 5b show the results of the PCR. Figures 6A and 6B provide the calculated percent recovery based on the number of copies that were spiked into the serum.

[00185] As indicated in Figures 5B and 6B, for DNA targets, the fully TamiSolve™- replaced workflow yields equivalently high sensitive DNA target isolation as that of standard alcohol-based workflows (extraction # 7 in 120bp DNA model graph).

[00186] For RNA targets, a slightly less optimal isolation was achieved with a fully TamiSolve™ replaced workflow (extraction #7 in MS2 RNA graph, Figure 5A). However, the use of TamiSolve™ for triggering binding and in wash buffer 1, while using 2- butoxyethanol in Wash buffer 2, results in an even better RNA recovery (extraction #6; Figures 5A and 6B).

Claims

2. The purification buffer of claim 1 , wherein the polar aprotic solvent is present in an amount of at least about 2 %wt; optionally wherein the polar aprotic solvent is present in an amount of not more than about 80 %wt.

3. The purification buffer of any preceding claim, wherein the polar aprotic solvent has a boiling point of more than 100 °C at 1 atm, optionally a boiling point of more than 150°C at 1 atm.

4. The purification buffer of any preceding claim, wherein the polar aprotic solvent comprises 2, 3 or 4 heteroatoms selected from O and N; and/or wherein the polar aprotic solvent comprises 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

5. The purification buffer of any preceding claim, wherein the polar aprotic solvent consists of carbon atoms, hydrogen atoms and 2, 3 or 4 heteroatoms selected from O and N.

6. The purification buffer of any preceding claim, wherein the polar aprotic solvent comprises 6, 7, 8, 9 or 10 carbon atoms.

7. The purification buffer of any proceeding claim, wherein the polar aprotic solvent has a viscosity of less than 50 cP at 20 °C and 1 atm; optionally wherein the polar aprotic solvent has a viscosity of less than 25 cP at 20 °C and 1 atm.

8. The purification buffer of any proceeding claim, wherein the polar aprotic solvent has a flashpoint of at least 50 °C; and/or wherein the polar aprotic solvent is liquid at 0 °C and 1 atm.

9. The purification buffer of any preceding claim, wherein the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine.

10. The purification buffer of any preceding claim, wherein the purification buffer further comprises one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof.

11. The purification buffer of any preceding claim, wherein the purification buffer is a binding buffer and/or a wash buffer; optionally wherein the wash buffer comprises a 1.1 to 5 times dilution of the binding buffer.

12. The purification buffer of any preceding claim, wherein the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or dimethylsulfoxide.

13. The use of a purification buffer of any of claims 1 to 12 to precipitate a nucleic acid from solution to a solid support, thereby providing a nucleic acid bound solid support.

14. The use of claim 13, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties.

15. The use of claim 13 or 14, wherein the solid support comprises one or more of particles, resin, cartridge, beads, a filter, a column, an array, a membrane, a chip, a disc or a slide.

16. The use of any of claims 13 to 15, wherein the solid support comprises (optionally monodisperse) beads; further optionally wherein the (optionally monodisperse) beads are magnetic.

17. The use of any of claims 13 to 16, further comprising washing the nucleic acid bound solid support with a wash buffer; optionally wherein the wash buffer is or comprises the purification buffer.

18. The use of any of claims 13 to 17, further comprising contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support; optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.

19. The use of claim 18, wherein the separated nucleic acid is further subjected to one or more additional processes, optionally selected from detection, cloning, restriction, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

20. The use of any of claims 13 to 19, wherein the nucleic acid is DNA; optionally one or more of synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA; or wherein the nucleic acid is RNA; optionally one or more of mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).

21. A method for processing a nucleic acid, the method comprising: exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support.

22. The method of claim 21 , wherein the aqueous medium comprising the polar aprotic solvent is an aqueous nucleic acid purification buffer as defined in any of claims 1 to 12.

23. The method of claim 21 or claim 22, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties; and/or wherein the solid support comprises one or more of particles, resin, cartridge, beads, a filter, a column, an array, a membrane or a chip; optionally wherein the solid support comprises (optionally monodisperse) beads.

24. The method of any of claims 21 to 23, further comprising washing the nucleic acid bound solid support with a wash buffer; optionally wherein the wash buffer is or comprises the purification buffer; and/or further comprising contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid.

25. The method of any of claims 21 to 24, wherein the nucleic acid is further subjected to one or more additional processes, optionally selected from detection, cloning, nucleic acid synthesis and/or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, protein expression, hybridisation, cDNA synthesis, size separation, chromatography, mass spectrometry, pharmaceutical or therapeutic formulation, and genome editing.

26. The method of any of claims 21 to 25, wherein the sample comprises or is a pre-treated or untreated biological sample, environmental sample, or an enzymatic reaction mixture.

27. The method of any of claims 21 to 26, further comprising performing point-of-care detection; optionally wherein the point-of-care detection comprises pathogen detection, biological warfare agent detection or genetic disease detection.

28. A kit comprising an aqueous nucleic acid purification buffer as defined in any of claims 1 to 12, and a solid support.

29. The kit of claim 28, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties; and I or wherein the solid support comprises one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane or a chip; optionally wherein the solid support comprises (optionally monodisperse) beads; and I or wherein the kit further comprises one or more of a lysis buffer, a wash buffer, and an elution buffer; optionally wherein the lysis buffer and/or the wash buffer comprise the polar aprotic solvent of the aqueous nucleic acid purification buffer.

30. A composition of matter comprising:

(i) a solution comprising an aqueous nucleic acid purification buffer as defined in any of claims 1 to 12; and

(ii) a nucleic acid solid support.

31 . The composition of claim 30, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise part of silanol, carboxyl, or saccharide moieties; and I or wherein the solid support comprises one or more of particles, resin, beads, a filter, a column, a cartridge, an array, a membrane or a chip; optionally wherein the solid support comprises (optionally) monodisperse beads.

32. Use of a kit of claim 28 or claim 29 in an automated nucleic acid analysis platform.

33. A nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of claim 28 or claim 29, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.

34. The nucleic acid analysis apparatus of claim 33, wherein the automated nucleic acid analysis platform is a point-of care assay instrument; optionally wherein the point-of-care assay instrument is adapted for pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection.