WO2025078670A1 - Sequential nucleic acid isolation - Google Patents

Abstract

Provided herein is a method of sequentially isolating DNA and RNA from a sample, the method comprising: contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA-bound solid support; removing the DNA-bound solid support from the sample; contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent. Also provided is a kit comprising said purification buffer and solid supports, the use of said kit in an automated nucleic acid analysis platform, and a nucleic acid analysis apparatus, comprising an automated nucleic acid analysis platform; and the aforementioned kit.

Description

Sequential Nucleic Acid Isolation

[0001] This invention relates to a method of sequentially isolating DNA and RNA from a sample, as well as kits and apparatus relating to the same. The method involves binding the DNA and RNA to different solid supports. 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] When extracting rare targets such as tumor-derived analytes such as circulating tumor cells (CTC), tumor derived proteins, ctDNA and ctRNA for multiomics based analysis, it is vital to capture as much as possible of the available analyte in the sample matrix. As current multiomics analysis require different readout methods depending on the analyte (e.g. mass spectrometry or immunoassay for proteomics, NGS for genomics and epigenomics and transcriptomics), protein, DNA and RNA targes need to be extracted separately to enable the different analysis workstreams. Currently different extraction chemistry and solid phases are required for the extraction of protein versus nucleic acids. There is, however, a big overlap between the extraction of DNA versus RNA, which makes it especially challenging to efficiently separate these two. Currently, this challenge is partially solved by changing the lysis binding buffer chemistry between the DNA and RNA extraction. Unfortunately the specific isolation of only DNA versus RNA targets is challenging due to: high carry-over of respective targets despite modifying the chemistry between RNA and DNA extraction causing loss of precious RNA or DNA targets; changing buffer composition which requires adding additional buffer components to an already complex workflow; and being mostly based on alcohol or polyol-based precipitation of nucleic acids which makes the chemistry either flammable (i.e. alcohol based - hence not suitable for POC cartridges) or highly viscous (polyol-based, hence challenging to automate on liquid handlers). Changing the buffer composition in such a manner will also dilute the original sample and thereby the molecules to be isolated even further. A protein isolation prior to isolation of nucleic acids will enhance this effect.

[0003] For Qiagen’s AHPrep® DNA/RNA/Protein a lysate is passed through an AHPrep DNA spin column, which, in combination with a high-salt buffer, allows selective binding of genomic DNA. The column is then washed, and the bound DNA is eluted. Ethanol is then added to the flow-through from the spin column to provide appropriate binding conditions for RNA. The sample is applied to an RNeasy spin column where total RNA binds to the membrane. Qiagen’s AHPrep® DNA/RNA/Protein Kit therefore requires both the lysis binding buffer chemistry and the solid phase to be changed in order to facilitate sequential DNA and RNA extraction. [0004] For Cytiva’s triplePrep Kit chaotropic salt in Lysis buffer type 15 promotes the binding of DNA to a silica membrane. The DNA-bound silica membrane is then washed, and the DNA is eluted. Acetone is then added to the flow-through which promotes the binding of total RNA to the silica membrane in the presence of chaotropic salt in Lysis buffer type 15. Cytiva’s triplePrep Kit therefore requires the lysis binding buffer chemistry to be changed in order to facilitate sequential DNA and RNA extraction.

[0005] There is therefore a need for improved methods of sequentially isolating DNA, RNA and optionally, protein, from the same sample in an efficient manner. This is particularly relevant where there is only a modest amount of sample and/or a low concentration of one or more of the target molecules within the sample, so strategies that involve splitting and/or diluting the sample may result in amounts of isolated molecules that are below detection limits. In addition, such improved methods may enable efficient high- throughput automation to POC cartridge-based automation of sample processing for multiomics readout for early cancer diagnostics, early neurological disease diagnostics, precision medicine, and treatment response monitoring.

BRIEF SUMMARY OF THE DISCLOSURE

[0006] A first aspect of the invention provides a method of sequentially isolating DNA and RNA from a sample, the method comprising steps a) to d): a) contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA- bound solid support; b) removing the DNA-bound solid support from the sample; c) contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and d) removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.

[0007] 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.

[0008] 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.

[0009] 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 atoms. The polar aprotic solvent may comprise 1 or 2 O atoms and 1 or 2 N atoms. The polar aprotic solvent may comprise 2 O atoms. The polar aprotic solvent may comprise 3 O atoms. The polar aprotic solvent may comprise 1 O atom and 1 or 2 N atoms. The O atoms may comprise =0 and/or -O-.

[0010] 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.

[0011] 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.

[0012] The polar aprotic solvent may have a flashpoint of at least 50 °C.

[0013] The polar aprotic solvent may be liquid at 0 °C and 1 atm.

[0014] The polar aprotic solvent may be non-flammable, non-viscous, EHS-friendly, and/or biorenewable.

[0015] The polar aprotic solvent may be an environmental, health and safety (EHS) friendly solvent.

[0016] 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. In preferred embodiments, the polar aprotic solvent is N-butylpyrrolidin-2-one.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Where the sample is not a liquid sample (i.e. a solid sample), the method may initially comprise suspending or dissolving the solid sample in a suitable buffer, e.g. the aqueous nucleic acid purification buffer.

[0025] The method may be a method of sequentially isolating protein, DNA and RNA from the sample, the method further comprising isolating protein before performing step a).

[0026] Isolating protein may be targeted or non-targeted separation. One or more targeted protein separations may be followed by one or more non-targeted protein separations.

[0027] Isolating protein from the sample may comprise a targeted affinity protein separation, such as immunoprecipitation. An affinity protein separation is conveniently carried out by any appropriate method using a solid phase with appropriate surface properties. Appropriate methods will depend on the type of target proteins. In one embodiment specific proteins may be isolated using a solid support, which has an appropriate binding partner/ ligand attached to its surface. This thus applies well known principles of affinity separation of proteins, as widely described in the prior art. Any such standard and well known methods may be used or adapted for the present invention.

[0028] The targeted affinity protein separation may comprises contacting the sample with a specific protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample. The method may comprise contacting the sample with the immobilized protein affinity reagent in the aqueous nucleic acid purification buffer that is used for the subsequent sequential isolation of DNA and RNA. The protein affinity reagent may be any molecule having an affinity for the target protein, e.g. an antigen, antibody, lectin, carbohydrate, metal ion(s), engineered protein scaffold, a substrate and/or an inhibitor of the target protein. Affinity reagents are well known to the skilled person, may be available commercially or produced individually, e.g. by using an antibody coupling kit which enables the user to covalently couple antibodies or antigens to a solid support, such as Dynabeads™ magnetic beads, for the use in immunoprecipitation workflows.

[0029] Targeted affinity protein separation (e.g. immunoprecipitation) typically requires diluting the sample in an appropriate phosphate buffer, such as a PBS or PBST buffer. One or more, e.g. two, three, four, etc. targeted affinity protein separations may be performed either consecutively or concurrently. When performing more than one targeted affinity separation using beads of different sizes or labelled differently may allow distinguishing the different separated proteins if required.

[0030] Alternatively, or subsequently, a solid phase with more general surface binding properties can be selected, e.g. a solid phase which has surface chemistry which effects classical chromatographic interactions such as ion exchange (including both anion exchange and cation exchange), reverse phase interactions, or hydrophobic interactions. Such surfaces may be conveniently provided on magnetic beads. The use of these more general surfaces conveniently allows the fractionation of proteins in the sample into subsets depending on the structure and properties (charge, hydrophobicity, etc.) of the proteins present. Some of these generic protein enrichment methods use weak to strong anionic or cationic surface chemistry. Such general solid phase surfaces can be prepared using conventional techniques which are standard and well documented in the art of column chromatography. Therefore, isolating protein from the sample may, e.g., comprise contacting the sample with a solid support comprising ionizable surface groups, lowering the pH of the sample to ionize the surface groups thereby causing the ionized surface groups to selectively bind both DNA and RNA to form a nucleic acid-bound solid support. The nucleic acid-bound solid support may then be removed from the protein, optionally washed (e.g. with a wash buffer) and contacted with an elution buffer, thereby separating the DNA and RNA from the solid support. In another embodiment SAX (strong anionic exchange) is used wherein negatively charged proteins or exosomes/viruses bind under physiological or low salt conditions to the solid support. A high salt (e.g. 1M NaCI) can then used to elute proteins from the support. The solution containing the DNA and RNA that did not bind to the solid support will form the (protein free) sample used in step a). This sample will typically be added to the aqueous nucleic acid purification buffer or vice versa.

[0031] The method may further comprise a lysis step. If necessary, e.g. if the nucleic acid and protein to be isolated is not available for binding within the initial sample (e.g. is contained within a biological particle such as a viral coat or a cell membrane or wall), the initial binding step may be preceded by one or more separate steps to free the nucleic acid and protein components by, e.g. disrupting structural components such as cell walls to achieve lysis. Procedures for achieving this are well known in the art. Thus, for example, although some cells e.g. blood cells, may be lysed by reagents such as detergent alone, other cells, e.g. plant or fungal cells or solid animal tissues may require more vigorous treatment such as, for example, grinding in liquid nitrogen, heating in the presence of detergent, alkaline lysis in the presence of detergent or freeze/thawing cycles. In many instances procedures for freeing nucleic acid and protein are chosen such that the particular nucleic acid and protein species which are to be isolated in the methods of the invention remain sufficiently intact, e.g. are not substantially degraded and the lysis condition does not negatively affect any antibody or antigen or their affinity for each other if an affinity method is used in the optional protein isolation step.

[0032] Typically, any lysis step is performed before step a) or during step a). It may be that the lysis step is performed before isolating protein from the sample. It may be that the lysis step is performed after isolating protein from the sample. A lysis step may also be performed between the targeted isolation of two or more different proteins or between a targeted and a more general protein isolation.

[0033] The lysis step may comprise adding a lysis buffer to the sample. After combining sample and lysis buffer additional lysis steps may be performed, like agitation, heating and/or incubation. It may be that the lysis buffer is the aqueous nucleic acid purification buffer. For certain samples it is possible to combine the lysis step with the binding step a) by adding the polar aprotic solvent and the solid support comprising surface silanol groups to the lysis buffer and provide a one-step lysis and binding workflow. The polar aprotic solvent and the solid support comprising surface silanol groups may be added prior to combining sample and lysis buffer or after combining sample and lysis buffer and optional additional lysis steps like agitation and/or incubation.

[0034] The lysis or lysis/nucleic acid purification buffer may comprise a high concentration of chaotrope (for example a guanidinium salt or urea) and surfactant, e.g. Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, Octyl-beta- Glucoside, Octylthio Glucoside, SDS (sodium dodecyl sulfate), CHAPS, and/or CHAPSO, preferably Tween-20. 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 Proteinase K), and the like.

[0035] The method may further comprise contacting the sample with a proteolytic enzyme, e.g. Proteinase K, prior to step a), provided that said proteolytic enzyme/Proteinase K is not added until after the (optional) protein isolation step. It may be that the method further comprises contacting the sample with Proteinase K and a lysis buffer prior to step a), provided that said Proteinase K and lysis buffer are not added until after the (optional) protein separation.

[0036] Typically, Steps a) and b) are carried out before steps c) and d). It may be however that steps c) and d) are carried out before steps a) and b).

[0037] In the contacting step a) the different components, such as sample, appropriate buffer, solid support comprising surface silanol groups and the polar aprotic solvent, may be added in any possible order. The method may therefore comprise adding the solid support comprising surface silanol groups to the sample which has been mixed with an appropriate buffer and adding the polar aprotic solvent as the last component. It is also possible to, e.g., mix an appropriate buffer with the polar aprotic solvent first, then add the sample and finally the solid support. It may be that the aqueous nucleic acid purification buffer comprising the polar aprotic solvent and the solid support comprising surface silanol groups are added to the sample sequentially or at the same time, e.g. the aqueous nucleic acid purification buffer may contain the solid support comprising surface silanol groups dispersed therein. It may be that an excess amount of solid support comprising surface silanol groups is added to the sample.

[0038] Typically, the method will comprise adding the solid support comprising surface carboxyl groups to the sample after step b).

[0039] Step b) may further comprise optional step b2): washing the DNA-bound solid support with a wash buffer. Step b2) may comprise washing the DNA-bound solid support at least once. Step b2) may comprise washing the DNA-bound solid support at least twice, e.g. at least three times, each time with fresh wash buffer. It may be that the wash buffer comprises the purification buffer. 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 neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.

[0040] 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.

[0041] Step b2) may comprise washing the DNA-bound solid support with wash buffer 1. Wash buffer 1 may comprises a nucleic acid purification buffer. It may be that wash buffer 1 comprises at least one polar aprotic solvent (e.g., the polar aprotic solvent that is present in the aqueous nucleic acid purification buffer, such as dihydrolevoglucosenone, N- butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N-Formylmorpholine; e.g. N- butylpyrrolidin-2-one). Wash buffer 1 may comprise from about 25 to about 75% by volume the polar aprotic solvent, such as dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N-Formylmorpholine; e.g. N-butylpyrrolidin-2-one. Wash buffer 1 may comprise from about 35 to about 65% by volume the polar aprotic solvent, such as dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N-Formylmorpholine; e.g. N-butylpyrrolidin-2-one. Wash buffer 1 may comprise from about 45 to about 55% by volume the polar aprotic solvent such as dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N- Formylmorpholine; e.g. N-butylpyrrolidin-2-one.

[0042] Wash buffer 1 may comprise a polar organic solvent (other than, or in addition to, the polar aprotic solvent that is present in the aqueous nucleic acid purification buffer). The polar organic solvent may be an alcohol, e.g. a Ci-Ce alcohol, polyol, or oligoethylene glycol. The polar organic solvent may be Ci-Ce alcohol. The polar organic solvent may be isopropyl alcohol.

[0043] Wash buffer 1 may comprise from about 25 to about 75% by volume the organic solvent (e.g., isopropyl alcohol). Wash buffer 1 may comprise from about 35 to about 65% by volume the organic solvent (e.g., isopropyl alcohol). Wash buffer 1 may comprise from about 45 to about 55% by volume the organic solvent (e.g., isopropyl alcohol).

[0044] Step b2) may comprise washing the DNA-bound solid support with wash buffer 2. It may be that wash buffer 2 does not comprises a nucleic acid purification buffer. It may be that wash buffer 2 comprises an organic solvent (e.g. a polar organic solvent) and, optionally, water. Wash buffer 2 may comprise greater than, or equal to, about 25% by volume the organic solvent. Wash buffer 2 may comprise greater than, or equal to, about 30%, e.g. about 35%, by volume the organic solvent. Wash buffer 2 may comprise less than, or equal to, about 90% by volume the organic solvent. Wash buffer 2 may comprise less than, or equal to, about 80% by volume the organic solvent. It may be that at least some (e.g. all) of the remaining wash buffer 2 volume is water.

[0045] The organic solvent may be an alcohol, e.g. a Ci-Ce alcohol, diol (e.g. methylpropyl diol), polyol, or oligoethylene glycol. It may be that the organic solvent is a Ci-Ce alcohol. The alcohol may be ethanol, or isopropanol. Wash buffer 2 may comprise from about 50 to about 90% by volume of an alcohol (e.g., ethanol). Wash buffer 2 may comprise from about 60 to about 80% by volume of an alcohol (e.g., ethanol). It may be that the organic solvent is a glycol ether, e.g. a C2-C10 glycol ether. The glycol ether may be 2-butoxyethanol. Wash buffer 2 may comprise from about 25 to about 75% by volume of a glycol ether (e.g., 2-butoxyethanol). Wash buffer 2 may comprise from about 35 to about 65% by volume of a glycol ether (e.g., 2-butoxyethanol).

[0046] It may be that wash buffer 2 comprises less than about 50% by volume N- butylpyrrolidin-2-one. It may be that wash buffer 2 comprises less than about 25% by volume N-butylpyrrolidin-2-one. It may be that wash buffer 2 comprises less than about 5% by volume N-butylpyrrolidin-2-one. It may be that wash buffer 2 does not comprise N- butylpyrrolidin-2-one.

[0047] Step b2) may comprise washing the DNA-bound solid support with at least two different wash buffers. For example, step b2) may comprise washing the DNA-bound solid with wash buffer 1 and wash buffer 2, wherein wash buffers 1 and 2 are different. It may be that step b2) comprises washing the DNA-bound solid support with wash buffer 1 before washing the DNA-bound solid support with wash buffer 2.

[0048] Step b) may further comprise optional step b3): contacting the DNA-bound solid support with an elution buffer, thereby separating the DNA from its solid support. It may be that the elution buffer comprises 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.

[0049] Step d) may further comprise optional step d2): washing the RNA-bound solid support with a wash buffer. Step d2) may comprise washing the RNA-bound solid support at least once. Step d2) may comprise washing the RNA-bound solid support at least twice, e.g. at least three times, each time with fresh wash buffer. It may be that the wash buffer is or comprises the purification buffer. 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 neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.

[0050] 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.

[0051] Step d2) may comprise washing the RNA-bound solid support with at least two different wash buffers. For example, step d2) may comprise washing the RNA-bound solid with wash buffer 1 and wash buffer 2, wherein wash buffers 1 and 2 are different. It may be that step d2) comprises washing the RNA-bound solid support with wash buffer 1 before washing the RNA-bound solid support with wash buffer 2. Wash buffers 1 and 2 may be as defined above in connection with step b2).

[0052] Step d) may further comprise optional step d3): contacting the RNA-bound solid support with an elution buffer, thereby separating the RNA from its solid support. It may be that the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof. A conventional elution buffer may be used, such as for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 mM Tris-HCI buffer at pH 8.0.

[0053] In embodiments, the method comprises contacting the sample with a protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample. The method may comprise contacting the sample with the immobilized protein affinity reagent in the aqueous nucleic acid purification buffer that is used for the subsequent sequential isolation of DNA and RNA. In these embodiments, the method may further comprise washing the protein-bound solid support with a wash buffer. For example, the method may comprise washing the protein-bound solid support at least once. The method may comprise washing the proteinbound solid support at least twice, e.g. at least three times, each time with fresh wash buffer. It may be that the wash buffer is or comprises a physiological buffer, PBS, HEPES, Tris, NH4OAC, etc with or without salts (e.g. KOAc, NaCI, MgCh, KOI), with or without ionic or non-ionic, anionic or cationic, or zwitterionic detergents, with or without reducing agents, with or without EDTA/EGTA etc., depending on the nature of the affinity reagent and the target protein as well as subsequent uses of the isolated target protein.

[0054] The method may further comprise contacting the protein-bound solid support with an elution buffer, thereby separating the protein from its solid support. It may be that the elution buffer comprises an aqueous salt solution, such as saline, or solution comprising glycine-HCI at a pH of around 2.5-3.0.

[0055] The separated protein, DNA and/or RNA may be further subjected to one or more additional processes like identification and/or quantification. These additional downstream processes may be selected from but not limited to e.g. microarray analysis, detection, cloning, restriction, nucleic acid synthesis and/or assembly, epigenetic analysis, sequencing, amplification, transfection, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

[0056] The amplification may comprise PGR, qPCR, digital PGR (dPCR), reverse transcription, in vitro transcription, or isothermal amplification. The isothermal amplification may comprise 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) (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).

[0057] 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 Applied Biosystems, Thermo Fisher Scientific), 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.

[0058] It may be that the separated DNA is further subjected to one or more additional processes selected from but not limited to, e.g. microarray analysis, qPCR, dPCR and next-generation sequencing.

[0059] It may be that the separated RNA is further subjected to one or more additional processes selected from but not limited to e.g. reverse transcription, microarray analysis, qPCR, dPCR and next-generation sequencing.

[0060] The processes of the present invention can also be used for extraction and subsequent analysis of fragmented DNA.

[0061] The method may further comprise performing point-of-care (POC) detection. The point-of-care detection may comprise e.g. testing for clinical markers or drugs, 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 and/or protein detection. The POC detection may be a single or a multiplex detection. The testing for clinical markers or drugs as well as the genetic disease detection may include prenatal testing.

[0062] Any isolated protein(s) may be further subjected to one or more additional processes. After a targeted affinity separation, the protein may be quantified. After a non targeted separation proteins may be further separated, identified and/or quantified using methods well known in the art. The one or more additional processes may be selected from e.g. protein microarray analysis, mass spectrometry, light scattering, immunoassays like e.g. ELISA or Western blot, protein sequencing, enzymatic activity and/or other methods known in the art.

[0063] The solid support comprising surface silanol groups 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 comprising surface silanol groups may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support comprising surface silanol groups may be a component of a robotic liquid handling platform.

[0064] The solid support comprising surface carboxyl groups 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 comprising surface carboxyl groups may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support comprising surface carboxyl groups may be a component of a robotic liquid handling platform.

[0065] The solid support to which the protein affinity reagent (e.g. antigen, ligand, antibody, peptide, aptamer, lectin, carbohydrate, metal ions, engineered protein scaffold, a substrate and/or an inhibitor or metabolite of the target protein is immobilized 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 to which the protein affinity reagent is immobilized may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support to which the protein affinity reagent is immobilized may be a component of a robotic liquid handling platform.

[0066] The solid support comprising ionizable surface groups 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 comprising ionizable surface groups may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support comprising ionizable surface groups may be a component of a robotic liquid handling platform.

[0067] The DNA may, for example, be one or more of synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA. The DNA may be ctDNA.

[0068] The RNA may, for example, be one or more of mRNA (e.g. isolated from a biological sample or in v/tro-transcribed), siRNA, microRNA, tRNA, cfRNA, rRNA, viral RNA (e.g. dsRNA or ssRNA), snRNA, or ctRNA. The RNA may be ctRNA.

Sample

[0069] The sample may comprise or be a pre-treated or untreated biological sample, clinical or 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.

[0070] The biological sample may be any suitable biological sample. Exemplary biological samples may comprise but are not limited to 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 (e.g. tumor 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, exosomes, circulating tumor cells, tissue lysates, bacterial lysates, yeast lysates, and plant lysates. The biological sample may comprise exosomes. The biological sample may be a fluid biological sample. In some instances, the biological sample may be a clinical sample. In some instances, the sample may be a cell-free sample.

[0071] The environmental sample may comprise a water sample (such as a wastewater sample, swimming pool water sample, ocean water 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.

[0072] 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.

[0073] The method of the present invention may advantageously allow for the sequentially extraction of RNA and DNA present in a sample at very low numbers in a large sample volume (e.g. cfDNA or cfRNA, ctDNA or ctRNA in plasma) or from small samples (e.g. single- or few-call samples, as is often the case with CTCs). It may therefore that the sample comprises DNA and/or RNA at levels ranging from about 1 picogram nucleic acid/mL (or even lower) to about 1 microgram of nucleic acid/mL (or even higher). qPCR or RTqPCR may be used for detection and quantification of DNA/RNA in the below picogram range. In the pico to nanogram range fluorescent nucleic acid intercalating dyes may be used for detection and quantification.

[0074] The specific DNA and RNA to be isolated will vary based on the sample. For example, the sample may comprise CTCs, the DNA may comprise gDNA and the RNA may comprise total RNA. It may be that the sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.

[0075] A second aspect of the invention provides a kit comprising: a first solid support comprising surface silanol groups; a second solid support comprising surface carboxyl groups; and an aqueous nucleic acid purification buffer comprising a polar aprotic solvent.

[0076] The kit may additionally comprise components for one or more targeted protein isolations and/or non-targeted protein isolations, such as a third solid support comprising an immobilized protein affinity reagent, such as e.g. a tosylactivated surface to bind an antibody directed at a target protein to be isolated or a solid support having a surface that is suitable to bind to a target protein, subgroup of proteins or all proteins in a sample.

[0077] The first and second solid supports may be as defined in the first aspect above.

[0078] The aqueous nucleic acid purification buffer and a polar aprotic solvent may be as defined in the first aspect above.

[0079] 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. The kit may comprise two different wash buffers (wash buffers 1 and 2). Wash buffer 1 may be as defined above in connection with the first aspect of the invention. Wash buffer 2 may be as defined above in connection with the first aspect of the invention.

[0080] A third aspect of the invention provides a use of a kit of the second aspect of the invention in a nucleic acid analysis platform.

[0081] A fourth aspect of the invention provides a nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of the second aspect of the invention, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.

[0082] 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, clinical markers or drugs 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. In preferred embodiment, the point-of-care assay instrument is adapted for cancer detection. The testing for clinical markers, pathogens or drugs as well as the genetic disease detection may include prenatal testing. [0083] 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

[0084] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 provides an exemplary schematic illustrating a general process for the sequential isolation of protein, DNA and RNA from a sample.

Figure 2 provides an exemplary schematic illustrating a more detailed process for the sequential isolation of protein, DNA and RNA from a liquid biopsy sample.

Figure 3 is a bar chart showing the recovery of nucleic acids from silane and carboxylic acid functionalised stationary phases using the aprotic solvent N- butylpyrrolidin-2-one (TamiSolve™) in the binding buffer, with the protic solvent isopropanol (I PA) used as a positive control.

DETAILED DESCRIPTION

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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

[0090] 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.

[0091] 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).

[0092] 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 to a solid support. Exemplary purification buffers may therefore also be considered binding buffers or washing buffers.

[0093] 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.

[0094] 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.

[0095] The term “elution buffer” means, unless the context requires otherwise, a buffer that is useful for eluting (removing) a substance, e.g. nucleic acid or protein, from a solid support (to which the substance is bound). The elution buffer to be used will depend on the substance to be eluted and the solid support. For example, a nucleic acid elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof. A protein elution buffer on the other hand may comprise an aqueous salt solution, such as saline, a solution comprising glycine-HCI at a pH of aroundf 2.5-3.0, a solution comprising NH4OH and/or buffers suitable for protease digestion (e.g. trypsin digestion) of the eluted protein e.g. for subsequent mass spectrometry analysis.

[0096] Targeted protein isolation is specific to a particular protein, or class of protein. An example of targeted protein isolation is immunoprecipitation in which an antibody with a specific affinity for a protein of interest immobilized to a solid support (e.g. a bead) is contacted with the sample causing that protein to bind to the support (e.g. the bead).

[0097] Non-targeted protein isolation is not specific to a particular type of protein and aims to isolate a certain subgroup of proteins (e.g. all lipophilic proteins) or all proteins from a sample. An example of non-targeted protein isolation is contacting the sample with a solid support comprising silica in which the surface silanol groups or bound to alkyl chains of a certain length, e.g. C4, Cs or C18. In this example, the solid support may be the stationary phase of a column, e.g. a reverse phase column. The terms “protein isolation” and “protein separation” are used synonymously.

[0098] 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.

[0099] 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 %.

[00100] 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.

[00101] A solid support comprising surface silanol groups may be composed of comprise silica, which, at its surface, terminates in, inter alia, silanol groups ( -Si-OH).

[00102] Solid supports 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.

[00103] 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 (and patent publication No. WO 2024/018084 A2), which are hereby incorporated by reference, may be used. In some examples the solid support comprising surface silanol groups may be selected from any commercially available solid support suitable for binding nucleic acid comprising surface silanol groups. For example, the solid support comprising surface silanol groups may be selected from Dynabeads™ MyOne™ Silane, SeraSil-Mag 400 or 700 (Cytiva), Silicon Hydroxyl Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), and MagneSil™ beads (available from Promega).

[00104] In some examples the solid support comprising surface carboxyl groups may be selected from any commercially available solid support suitable for binding nucleic acid comprising surface carboxyl groups. The solid support comprising surface carboxyl groups may be selected from Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M- 270™ Carboxylic Acid (all available from Thermo Fisher Scientific), SeraMag SpeedBeads™ carboxylate-modified (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), Carboxyl 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), 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™ Carboxyl, 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.

[00105] In some examples the solid support used to immobilize the protein affinity reagent may be selected from any commercially available solid support suitable for binding protein. The solid support used to immobilize the protein affinity reagent may be selected from e.g. Dynabeads™ MyOne™ Epoxy, Dynabeads™ M-270 Epoxy beads, Dynabeads™ MyOne™ Tosylactivated and Dynabeads™ Protein A, Dynabeads™ Protein G, Dynabeads™ Protein A/Protein G and Dynabeads™ M-280 Streptavidin, Dynabeads M- 270 Streptavidin, Dynabeads MyOne Streptavidin C1, Dynabeads MyOne Streptavidin T1, Dynabeads™ Sheep-Anti Mouse IgG, Dynabeads™ M-280 Sheep Anti-Mouse IgG, Dynabeads™ M-280 Sheep Anti-Rabbit IgG, Dynabeads™ Sheep Anti-Rat IgG, Dynabeads™ Rat Anti-Mouse IgM, Dynabeads™ Goat Anti-Mouse IgG, Dynabeads™ Protein A for Immunoprecipitation, and DynaGreen™ CaptureSelect™ Anti-IgG-Fc (MultiSpecies) Magnetic Beads (all available from Thermo Fisher Scientific).

[00106] 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.

[00107] The term “glycol ether” means, unless stated otherwise, a compound comprising a hydroxyl group and an ether group. Exemplary glycol ethers include, but are not limited to, a C2-C10 glycol ether, where the glycol ether can be an alkane or alkene substituted by a hydroxyl group and interrupted by 1, 2 or 3 ether linkages, where chemically possible. Exemplary glycol ethers include 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, 2- isopropoxyethanol, 2-butoxyethanol, 1-methoxy-2-propanol, 2-(2-methoxyethoxy)ethanol, and 2-(2-ethoxyethoxy)ethanol.

[00108] 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.

[00109] 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).

[00110] The term “flash point” 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 flash point above 50 degrees Celsius would only be an ignition risk under specific extreme conditions. Flash points of some common solvents can be found in the following table.

[00111] Throughout the specification these abbreviations have the following meanings:

2-BE 2-butoxyethanol cDNA complementary DNA cfDNA cell-free DNA cfRNA cell-free RNA

CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1 -propanesulfonate)

CHAPSO 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1 -propanesulfonate CHES 2-(Cyclohexylamino)ethanesulfonic acid cP centipoise

CTCs circulating tumor cells ctDNA circulating tumor DNA ctRNA circulating tumor RNA

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid dPCR digital polymerase chain reaction dsDNA double Stranded DNA dsRNA double Stranded RNA

DTT dithiothreitol

EDTA ethylenediaminetetraacetic acid

EGTA ethyleneglycol- b/s(P-aminoethyl)-N,N,N',N'-tetraacetic Acid gDNA genomic DNA

HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid

I PA isopropanol

LPA linear polyacrylamide

MOPS 3-morpholin-4-ylpropane-1 -sulfonic acid mRNA messenger RNA

PBS phosphate buffered saline

PBST phosphate buffered Saline with Tween™ 20

PCR polymerase chain reaction

PEG polyethylene glycol

POC point-of-care qPCR quantitative polymerase chain reaction

RNA ribonucleic acid rRNA ribosomal RNA

RTqPCR quantitative reverse transcription polymerase chain reaction SDS sodium dodecyl sulfate siRNA mall interfering RNA snRNA small nuclear RNA ssRNA single-stranded RNA

TAPS N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid

TCEP tris(2-carboxyethyl)phosphine

TEG tetraethylene glycol tRNA transfer RNA

TS TamiSolve™

P-ME p-mercaptoethanol

Method

[00112] In Figure 1 the sample is initially subject to protein separation (e.g. immunoprecipitation) to isolate any protein in the sample. Any isolated protein can then be subject to various downstream processes. An aqueous nucleic acid purification buffer comprising a polar aprotic solvent and solid support (e.g. magnetic beads) comprising surface silanol groups are then added to the sample. The formed DNA-bound solid support is then washed and eluted to form free DNA which can then be subject to various downstream processes. Solid support (e.g. magnetic beads) comprising surface carboxyl groups is then added to the sample. The formed RNA-bound solid support is then removed, washed and eluted to form free RNA which can then be subject to various downstream processes.

[00113] Step 0 of Figure 2 (optional, not depicted): Enrichment of the source of protein and/or nucleic acid to be isolated using techniques known in the art, e.g. circulating tumor cell (CTC) or exosome isolation using e.g. Dynabeads™ magnetic beads coupled to CTC or exosome specific antibodies, or SAX/Charge Switch beads for generic exosome enrichment.

[00114] Step 1 of Figure 2: Protein isolation from liquid biopsy sample matrix cleared of cells. Alternatively, the protein may similarly be isolated from, for example, single cell or tumor-derived exosome lysates, or from any other source of protein and nucleic acids (e.g. plasma, serum, urine, etc.) suitable for immunoprecipitation of protein targets. The protein isolation from the sample is provided by adding target-specific antibody coupled beads directly into the sample matrix. Move bead/protein complex to a fresh tube for further processing until the captured protein is ready for downstream readout (e.g. mass spectrometry, immunoassay, etc). Instead of, or in addition to a targeted protein isolation a non-targeted protein isolation may be performed. It is also possible to isolate several different proteins by repeating the targeted isolation steps using solid supports with different affinities specific for the respective target, e.g. beads coupled to different target specific antibodies. Optionally, these sequential targeted protein isolations or the single targeted protein isolation may be followed by a non-targeted protein isolation to precipitate the remaining proteins. [00115] Step 2 of Figure 2: DNA isolation by adding ProK, Lysis buffer, Dynabeads™

MyOne™ Silane, and the aprotic solvent to trigger DNA to bead binding. Move bead/DNA complex to a fresh tube for downstream processing.

[00116] Step 3 of Figure 2: RNA isolation by adding Dynabeads™ MyOne™ Carboxylic acid to the DNA-cleared lysate for RNA binding. Remove sample matrix, and continue processing.

[00117] Step 4 of Figure 2: RNA isolation by adding Dynabeads™ MyOne™ Carboxylic acid to the DNA-cleared lysate for RNA binding. Remove sample matrix, and continue processing.

Buffers and Solvents

[00118] Methods for isolating 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.

[00119] The nucleic acid purification buffer comprising a polar aprotic solvent utilised in the method of the present invention provides a number of advantages when compared to purification buffers used in the prior art, which are based on, e.g. alcohols and polyols. The inventors have shown that purification buffers comprising a polar aprotic solvent may promote differential binding of DNA and RNA to solid supports (e.g. beads) functionalized with silanol and carboxyl groups, respectively, from the same sample, without the need to change the purification buffer. This greatly simplifies and reduces the complexity and cost of the total DNA, RNA and, optionally, protein sequential isolation workflow for multiomics readout. Furthermore, 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 variation 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.

[00120] Biorenewable solvents are sourced from renewable, sustainable biobased materials, significantly lowering their environmental impact. Biorenewable solvents may, for example, be >50 % biobased through either carbon testing, or tracing the biobased content at all steps of manufacturing.

[00121] 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.

[00122] The polar aprotic solvent may be or comprise N-butylpyrrolidin-2-one (Tamisolve™):

[00123] The polar aprotic solvent may be or comprise dihydrolevoglucosenone (Cyrene™):

[00124] The polar aprotic solvent may be or comprise dipropylene glycol dimethyl ether (Proglyde™):

[00125] 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™, Brij®, 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.

[00126] The terms nucleic acid purification buffer and (nucleic acid) binding buffer may be used interchangeably.

[00127] 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. Any suitable combination of chaotropic agents 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. In some embodiments, the chaotropic agent is selected from a guanidinium salt (e.g. guanidinium isothiocyanate, guanidinium isocyanate or guanidinium hydrochloride), lithium perchlorate, lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, thiocyanate salts, 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 4M, e.g about 3-6M. In some embodiments, the lysis or lysis/binding buffer contains a guanidinium salt at a concentration of at least about 1M, at least about 1.5M, at least about 2M, at least about 3M, such as at least about 3.5M or about 4M, e.g. about 1-6M. In a preferred embodiment, the lysis buffer contains guanidinium (iso)thiocyanate at a final concentration of about 1.0- 4.5M, 1.5- 3.5M, or 3.5-4.5M, or guanidinium hydrochloride at a concentration of about 3.5- 4.5M, e.g. about 4.0M. The final concentrations during lysis and during binding steps differ due to addition of sample and trigger, respectively.

[00128] 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) polyoxyethylene-20-sorbitan monolaurate (Tween®- 20)™, Ecosurf™, CHAPS, Brij®, Zwittergent® 3-14 and a combination thereof. In a preferred embodiment, the detergent is selected from polyoxyethylene-20-sorbitan monolaurate (Tween®-20). 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 polyoxyethylene-20-sorbitan monolaurate (Tween®-20) at a concentration as defined above.

[00129] 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. polyoxyethylene-20-sorbitan monolaurate (Tween®-20). For example, a preferred lysis buffer is a high detergent, guanidinium isothiocyanate (GTC)-containing buffer.

[00130] 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. Instead of, or in addition to the one or more chelating agent one or more other inhibitors of DNases and/or RNases known in the art may be present in the lysis/binding buffer.

[00131] 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 (DTT), 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.

[00132] 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 1-20 mM, such as about 2-19 mM, 3-18 mM, 4-17 mM or about 5-16 mM, e.g. about 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 mM, preferably about 10 mM. However, in some embodiments, a higher amount of reducing agent may be used, e.g. about 20-150 mM, such as about 25-125 mM or about 30-100 mM, e.g about 80 mM.

[00133] 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.

[00134] 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.

[00135] 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).

[00136] 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.

Point-of-care Detection

[00137] 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.

[00138] 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 but are not limited to detection, quantification, cloning, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridisation, cDNA synthesis, size separation, chromatography, and mass spectrometry. The sample may be the supernatant of one or more targeted protein isolations and/or non-targeted protein isolation.

[00139] 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.

Automated nucleic acid analysis platforms

[00140] Automated nucleic acid analysis platforms have 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 protein and/or 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 KingFisher™ Systems, 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). The automated nucleic acid analysis platform may be a point-of care assay instrument.

[00141] Point-of-care assay instruments. The point-of-care assay instrument may be adapted for detection of clinical markers and/or drugs, and/or pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection. The pathogen detection or biological warfare agent detection may comprise viral and/or bacterial and/or fungal protein and/or 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. Currently available instruments also include cobas® 8000 modular analyzer series from Roche, the VIDAS® systems from bioMerieux or ARCHITECT analysers from Abbott. Siemens, DiaSorin and Ortho Clinical Diagnostics provide multi target analyser systems as well. KRYPTOR™ analysers are marketed by Thermo Fisher. 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

[00142] The invention and disclosure also comprise the subject matter of the following clauses:

1. A method of sequentially isolating DNA and RNA from a sample, the method comprising steps a) to d): a) contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA- bound solid support; b) removing the DNA-bound solid support from the sample; c) contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and d) removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.

2. The method of clause 1 , wherein the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine.

3. The method of clause 1 or clause 2, wherein the polar aprotic solvent is N- butylpyrrolidin-2-one.

4. The method of any preceding clause, further comprising adding the aqueous nucleic acid purification buffer to the sample prior to steps a) to d).

5. The method of any preceding clause, wherein the method is a method of sequentially isolating protein, DNA and RNA from the sample, the method further comprising isolating protein before performing step a).

6. The method of clause 5, wherein isolating protein comprises a non-targeted protein separation and/or an affinity protein separation, optionally wherein the non-targeted affinity protein separation comprises contacting the sample with a solid phase having a weak to strong anionic or cationic surface chemistry (which effects ion exchange), further optionally wherein the affinity protein separation is immunoprecipitation.

7. The method of clause 6, wherein the affinity protein separation comprises: contacting the sample with a protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample.

8. The method of any preceding clause, wherein the method further comprises a lysis step.

9. The method of clause 8, wherein the lysis step is performed before step a) or during step a); optionally wherein the lysis step is performed before affinity protein separation. The method of clause 8 or 9, wherein the lysis step comprises adding a lysis buffer to the sample. The method of any preceding clause, further comprising contacting the sample with Proteinase K prior to step a), provided that said Proteinase K is not added until after the (optional) affinity protein separation. The method of any preceding clause, wherein the solid supports comprise (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic. The method of any preceding clause, wherein step b) further comprises b2) washing the DNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer. The method of any preceding clause, wherein step b) further comprises b3) contacting the DNA-bound solid support with an elution buffer, thereby separating the DNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof. The method of clause 14, wherein the separated DNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and next-generation sequencing The method of any preceding clause, wherein step d) further comprises d2) washing the RNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer. The method of any preceding clause, wherein step d) further comprises d3) contacting the RNA-bound solid support with an elution buffer, thereby separating the RNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof. The method of clause 17, wherein the separated RNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and next-generation sequencing. The method of any preceding clause, 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. The method 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. 21. The method of any preceding clause, 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.

22. The method of any preceding clause, wherein the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, acetone, or dimethylsulfoxide.

23. The method of any preceding clause, wherein the DNA is cfDNA and/or ctDNA and/or wherein the RNA is cfRNA and/or ctRNA.

24. The method of any preceding clause, wherein the sample is a biological or environmental sample.

25. The method of any proceeding clause wherein the sample is a biological sample, optionally wherein: the biological sample is a fluid biological sample (e.g. a liquid biopsy); the biological sample is a cell based sample; the biological sample is single cell-based sample, CTC, exosomes, tissue biopsy material or FFPE; the biological sample comprise CTCs, the DNA comprise gDNA and the RNA comprise total RNA; or the biological sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.

26. A kit comprising: a first solid support comprising surface silanol groups; a second solid support comprising surface carboxyl groups; and an aqueous nucleic acid purification buffer comprising a polar aprotic solvent.

27. The kit of clause 26, wherein the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine, optionally wherein the polar aprotic solvent is N- butylpyrrolidin-2-one.

28. The kit of clause 26 or 27 further comprising a third solid support comprising an immobilized protein affinity reagent.

29. The kit of any one of clauses 26 to 28 further comprising a lysis buffer.

30. The kit of any one of clauses 26 to 29 further comprising Proteinase K. 31. The kit of any one of clauses 26 to 30, wherein the first solid support, and/or second solid support, and/or (optional) third solid support comprises (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.

32. The kit of any one of clauses 26 to 31 further comprising at least one wash buffer, optionally wherein the at least one wash buffer comprises the purification buffer.

33. The kit of any one of clauses 26 to 32 further comprising an elution buffer, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA, or a combination thereof.

34. The kit of any one of clauses 26 to 33, wherein the nucleic acid purification buffer further comprises the features defined in any of clauses 19 to 22.

35. Use of a kit of any one of clauses 26 to 34 in an automated nucleic acid analysis platform.

36. A nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of any one of clauses 26 to 34, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.

37. The nucleic acid analysis apparatus of clause 36, 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 cancer detection.

Example 1 : Protocol for the sequential extraction of circulating tumor cells or exosomes, and proteins, ctDNA and ctRNA from the same volume liquid biopsy sample

[00143] 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, a 120bp dsDNA fragment (1 e6 copies) and MS2 RNA (1.5 e6 copies) were spiked into 200pL human plasma prior to sample processing, and then processed according to the protocols outlined below. The eluted samples were then analyzed by qPCR to quantify the 120 bp DNA recovery or RT-qPCR to quantify the MS2 RNA recovery. The positive controls were processed using the same aqueous NA purification buffer with isopropanol instead of the polar aprotic solvent. Purifications were performed on a KingFisher instrument. [00144] Reagents:

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

• Carboxylic acid magnetic beads: Dynabeads™ MyOne™ Carboxylic Acid (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% IPA 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: 10 mM 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.

Table 1: Trigger parameters for Trigger testing

[00145] 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.

[00146] Procedure:

1. Protein isolation (optional): an exemplary workflow for an immunoprecipitation from liquid biopsies or soluble protein is provided in steps a) to g): a) Dilute the sample in 0.5 vol in PBS or PBST (pH 7.4) containing 5mM EDTA or a HEPES (pH 7.4) buffer with a detergents like Tween-20, such as those provided by the Dynabeads™ Co-Immunoprecipitation Kit (Thermo Fisher Scientific catalogue number 14321 D) b) Add your sample containing the antigen (Ag) (typically 100-1,000 pl) to the tube containing Dynabeads coupled Antibodies and gently pipette to resuspend the Dynabeads. c) Incubate with rotation for 10 minutes at room temperature to allow Ag to bind to the Dynabeads-Ab complex. Note: Depending on the affinity of the antibody, it may be necessary to increase incubation times for optimal binding. d) Place the tube on the magnet. Transfer the supernatant to a clean tube for further extraction of DNA/RNA or both (starting with an optional proteinase K digestion of step 3 below). e) Wash the Dynabeads-Ab-Ag complex three times using 200 - 500 pL Washing Buffer (e.g. PBST or an NH4OAc based buffer with or without detergent like those provided by the Dynabeads™ Co-Immunoprecipitation Kit (Thermo Fisher Scientific catalogue number 14321 D) depending on downstream analysis) for each wash. Separate on the magnet between each wash, remove supernatant and resuspend by gentle pipetting. f) Resuspend the Dynabeads-Ab-Ag complex in 100 - 500 pL Washing Buffer and transfer the bead suspension to a clean tube. This is recommended to avoid coelution of proteins bound to the tube wall, (at this point, the Ag bound to the beads can also be used in an immunoassay readout). g) Optionally elute the bound proteins in a desired volume of either 0.5M NH4OH with 0.5 mM EDTA (for Mass Spec analysis) or 200 mM Glycine (pH 2.8) for standard gel analysis.

2. Buffers were allowed to reach room temperature prior to use. Silane magnetic beads were vortexed for 20 seconds in order to re-suspend beads. Prior to use in step 6 the bottle was rotated on a roller/rotator at room temperature for 20 minutes.

3. 200 pL serum/plasma/UTM (Universal Transport Medium) sample or the supernatant of step 1 d) were added to a 1.5 mL tube.

4. 50 pL Proteinase K was added with a brief pulse-vortex.

5. 300 pL LB Buffer were added followed by a brief pulse-vortex and then incubated for 10 minutes at room temperature. In a thermal mixer/shaker, 10 min. incubation at 900 rpm would be sufficient.

6. 25 pL Dynabeads™ MyOne™ SILANE (1 mg) were added followed by a brief pulse-vortex.

7. 150 pL Trigger was gently added to the lysate and mixed with a brief pulse-vortex followed by incubation of the tube on a rotator/roller at room temperature for 10 minutes. In a thermal mixer/shaker, 10 min. incubation at 1050 rpm would be sufficient.

8. The tube was placed onto the DynaMag™-2 magnet. Beads were allowed to collect to the magnet for 1-2 minutes or until completely cleared.

9. Using a 1000 pL pipettor or a vacuum pump, the lysate was carefully removed. The lysate is then subject to step 25.

10. Wash Buffer 1 first wash: After removing the tube from the magnet, 800pl Wash Buffer 1 were added and vortexed until the beads were fully resuspended. The tube was placed onto the DynaMag™-2 magnet and beads were allowed to collect to the magnet for 1-2 minutes.

11. Using a 1000 pL pipettor or a vacuum pump, Wash Buffer 1 was carefully removed and discarded.

12. Wash Buffer 1 second wash: a second wash was performed by repeating steps 9-10 using the same volume of Wash Buffer 1 as in step 9.

13. Wash Buffer 2 first wash: after removing the tube from the magnet 500 pL Wash Buffer 2 were added and mixed by vortex/pipetting until the beads were fully resuspended. The bead solution was transferred using a 1000 pL pipettor to a fresh 1.5 mL microtube placed on the DynaMag™-2 magnet. 14. Beads were allowed to collect to the magnet for 1 minute. Wash Buffer 2 was carefully removed using a pipettor with a 1000 pL tip or a vacuum pump from the 1.5 mL tube and discarded.

15. Wash Buffer 2 second wash: Steps 13 and 14 were repeated (but without transferring the bead solution to a fresh tube).

16. To remove the trapped fluid between the beads, the rack/magnet with the tube was tapped down onto the bench top 3-5 times with medium force. Using a small pipette the final droplets were aspirated and discarded. All fluid was removed prior to elution.

17. 100 pL Elution Buffer were added.

18. After vortexing between 20 seconds and 1 minute until the bead pellet was fully resuspended and a brief centrifugation (1 second pulse) the bead suspension was collected. Care was taken not to pellet the beads.

19. The bead suspension was incubated in a pre-heated block at 80 °C for 5 minutes. In a thermal mixer/shaker, 5 min. at 80 °C mix with 1500 rpm would be sufficient.

20. After vortexing for 15 seconds the bead suspension was briefly centrifugated (1 second pulse).

21. The tube was placed on the DynaMag™-2 magnet. Beads were allowed to collect to the magnet for 15 seconds.

22. The eluted DNA was transferred t to a fresh 1.5 mL tube and stored at the appropriate temperature (e.g. 4 °C to -80 °C).

23. 100 pL Dynabeads™ MyOne™ Carboxylic Acid (1 mg) were added to the lysate from step 9 and mixed with a brief pulse-vortex.

24. The tube was incubated on a rotator/roller at room temperature for 10 minutes. When using a thermal mixer/shaker, 10 min. 900 rpm would be sufficient.

25. Steps 8 to 21 were performed on the 100 pL lysate containing Dynabeads MyOne Carboxylic Acid (1 mg).

28. The eluted RNA was transferred to a fresh 1.5 mL tube and stored at the appropriate temperature (e.g. 4 °C to -80 °C).

[00147] Results for TamiSolve™ are shown in Figure 3. When used with Silane Rare Target NA Purification Buffer, TamiSolve™ yields nearly 100% recovery of DNA targets while less than 4% of RNA targets bind to the silane beads. Subsequent binding of RNA to Dynabeads MyOne Carboxylic Acid yielded around 77% recovery of RNA targets.

Example 2: Wash buffer variation [00148] The protocol for the sequential extraction of DNA and RNA as described in Example 1 was repeated (positive control and Examples 2a-f). The trigger, wash buffer 1 and wash buffer 2 used in each repeat, as well as the DNA and RNA purification rates after the first and second elutions, respectively, are provided in Table 2, below. While this does not affect initial binding, final recovery can be influenced by the choice of wash buffer 1 and wash buffer 2.

Table 2: Variation of wash buffers

* volume 50% Silane Rare Target NA Purification Buffer

** volume water [00149] The data in Table 2 confirms that TamiSolve™ will trigger the binding of substantial amounts of DNA to Dynabeads™ MyOne™ SILANE and the binding of substantial amounts of RNA to Dynabeads™ MyOne™ Carboxylic Acid. The data also shows that good DNA and RNA purification rates can be achieved even when wash buffers 1 and 2 are varied. Example 3: Consistency for different silane beads

[00150] The protocol for the sequential extraction of DNA and RNA as described in Example 1 was repeated (positive control and Examples 3a-d). Two different batches of Dynabeads™ MyOne™ SILANE (bead batches a and b) were tested side by side. The trigger, wash buffer 1 and wash buffer 2 used in each repeat, as well as the DNA and RNA purification rates after the first and second elutions, respectively, are provided in Table 3, below. Similar results were obtained for both bead batches. Table 3: Comparison of results for two silane bead batches

** volume water

Example 4: Sequential protein, DNA and RNA purification

[00151] A plasma sample was spiked with 500 pg/mL IL-6 protein and 5.000.000 x 10⁶ copies/mL of 120bp DNA. 200 pl plasma with IL-6 and 120bp DNA was used per extraction and pipetted into a KingFisher plate. 20 pg Dynabeads™ MyOne™ Tosylactivated coupled with anti-IL-6 antibody were added, mixed slowly for 20 min, and placed on a magnet. The supernatant was aspirated and used for the isolation of nucleic acids. [00152] The beads were transferred into 200 pL Assay Buffer (TBST-BSA) and resuspended. 100 pL were transferred into white flat-bottom 96-well plate. 50 pL (0.1 pg/mL) acridinium ester (AE) labelled detection antibody were added and the plate was incubated on a shaker at 1000 rpm at 37 °C. Using a plate washer with a magnet the beads were washed 3x with 300 pL TBS. Finally, the beads were resuspended in 50 pL TBST and the signal was read on a Varioskan LUX using Flash. A standard curve for IL-6 was generated in parallel to quantify the amount of the precipitated protein.

[00153] The supernatant was subjected to DNA and RNA isolation as described in Example 1 after adding MS2 RNA (1.5 x 10⁶ copies).

[00154] The control workflow used I PA as a trigger for precipitation to the beads while the experimental workflows (Examples 4a and 4b) used Tamisolve as a trigger, but with varied wash buffer 2. The reagents used in each workflow, as well as the protein, DNA and RNA purification rates after each respective elution are provided in Table 4, below. Table 4: Protein, DNA and RNA purification rates

** volume water

[00155] The data in Table 4 shows that a polar aprotic solvent (e.g.TamiSolve™) can trigger the binding of substantial amounts of target protein to an appropriate solid support (e.g IL-6 antibody coupled to Dynabeads™ MyOne™ Tosylactivated) before triggering DNA and RNA binding in subsequent purifications without a change of buffer by a simple addition of an appropriate solid support (e.g. Dynabeads™ MyOne™ SILANE for DNA and Dynabeads™ MyOne™ Carboxylic Acid for RNA isolation).

Claims

1. A method of sequentially isolating DNA and RNA from a sample, the method comprising steps a) to d): a) contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA- bound solid support; b) removing the DNA-bound solid support from the sample; c) contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and d) removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.

2. The method of claim 1 , 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.

3. The method of claim 1 or claim 2, 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.

4. The method of any preceding claim, 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.

5. The method of any preceding 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.

6. The method of any preceding claim, wherein the polar aprotic solvent has a flash point of at least 50 °C.

7. The method of any preceding claim, wherein the polar aprotic solvent is liquid at 0 °C and 1 atm.

8. The method of claim 1 , wherein the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine.

9. The method of claim 8, wherein the polar aprotic solvent is N-butylpyrrolidin-2-one.

10. The method of any preceding claim, further comprising adding the aqueous nucleic acid purification buffer to the sample prior to steps a) to d).

11. The method of any preceding claim, wherein the method is a method of sequentially isolating protein, DNA and RNA from the sample, the method further comprising isolating protein before performing step a).

12. The method of claim 11 , wherein isolating protein comprises a non-targeted protein separation and/or an affinity protein separation, optionally wherein the non-targeted affinity protein separation comprises contacting the sample with a solid phase having a weak to strong anionic or cationic surface chemistry (which effects ion exchange), further optionally wherein the affinity protein separation is immunoprecipitation.

13. The method of claim 12, wherein the affinity protein separation comprises: contacting the fluid sample with a protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample.

14. The method of any preceding claim, wherein the method further comprises a lysis step.

15. The method of claim 14, wherein the lysis step is performed before step a) or during step a), optionally wherein the lysis step is performed before affinity protein separation.

16. The method of claim 14 or 15, wherein the lysis step comprises adding a lysis buffer to the sample.

17. The method of any preceding claim, further comprising contacting the sample with Proteinase K prior to step a), provided that said Proteinase K is not added until after the (optional) affinity protein separation.

18. The method of any preceding claim, 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.

19. The method of any preceding claim, wherein the solid support comprises (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.

20. The method of any preceding claim, wherein step b) further comprises b2) washing the DNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer.

21. The method of any preceding claim, wherein step b) further comprises b3) contacting the DNA-bound solid support with an elution buffer, thereby separating the DNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.

22. The method of claim 21, wherein the separated DNA is further subjected to one or more additional processes, optionally selected from detection, quantification, 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.

23. The method of claim 22, wherein the amplification comprises PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification.

24. The use of claim 23, 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).

25. The method of claim 22, wherein the separated DNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and nextgeneration sequencing.

26. The method of any preceding claim, wherein step d) further comprises d2) washing the RNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer.

27. The method of any preceding claim, wherein step d) further comprises d3) contacting the RNA-bound solid support with an elution buffer, thereby separating the RNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.

28. The method of claim 27, wherein the separated RNA 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, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

29. The method of claim 28, wherein the amplification comprises PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification.

30. The method of claim 29, wherein the isothermal amplification comprises 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).

31. The method of claim 27, wherein the separated RNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR, and nextgeneration sequencing.

32. The method of any preceding claim, wherein the polar aprotic solvent is present in an amount of at least about 2 %wt.

33. The method of any preceding claim, wherein the polar aprotic solvent is present in an amount of not more than about 80 %wt.

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

35. The method 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.

36. The method of any preceding claim, wherein the purification buffer is a binding buffer and/or a wash buffer.

37. The method of claim 36, wherein the wash buffer comprises a 1.1 to 5 times dilution of the binding buffer.

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

39. The method of any preceding claim, wherein the DNA is one or more of synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA.

40. The method of any preceding claim, wherein the RNA is one or more of mRNA (e.g. isolated from a biological sample or in v/tro-transcribed), siRNA, microRNA, tRNA, cfRNA, rRNA, viral RNA (e.g. dsRNA or ssRNA), snRNA, or ctRNA.

41. The method of any preceding claim, wherein the DNA is ctDNA and/or wherein the

RNA is ctRNA.

42. The method of any of any preceding claim, wherein the sample comprises or is a pre-treated or untreated biological sample, environmental sample, or an enzymatic reaction mixture.

43. The method of any preceding claim, wherein the sample is an environmental sample, optionally 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.

44. The method of any preceding claim, wherein the sample is an enzymatic reaction mixture, optionally 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.

45. The method of any preceding claim, wherein the sample is a biological sample, optionally 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.

46. The method of any proceeding claim, wherein the sample is a biological sample, optionally wherein: the biological sample is a fluid biological sample (e.g. a liquid biopsy); the biological sample is a cell based sample; the biological sample is single cell-based sample, CTC, exosomes, tissue biopsy material or FFPE; the biological sample comprise CTCs, the DNA comprise gDNA and the RNA comprise total RNA; or the biological sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.

47. A kit comprising: a first solid support comprising surface silanol groups; a second solid support comprising surface carboxyl groups; and an aqueous nucleic acid purification buffer comprising a polar aprotic solvent.

48. The kit of claim 47, wherein the polar aprotic solvent is as defined in any one of claims 2 to 9 and 32 to 34.

49. The kit of claim 47 or claim 48 further comprising a third solid support comprising an immobilized protein affinity reagent.

50. The kit of any one of claims 47 to 49 further comprising a lysis buffer.

51. The kit of any one of claims 47 to 50 further comprising Proteinase K.

52. The kit of any one of claims 47 to 51 , wherein the first solid support, and/or second solid support, and/or (optional) third solid support comprises (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.

53. The kit of any one of claims 47 to 52 further comprising at least one wash buffer, optionally wherein the at least one wash buffer comprises the purification buffer.

54. The kit of any one of claims 47 to 53 further comprising an elution buffer, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.

55. The kit of any one of claims 47 to 54, wherein the nucleic acid purification buffer is as defined in any of claims 35 to 36.

56. Use of a kit of any one of claims 47 to 55 in an automated nucleic acid analysis platform.

57. A nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of any one of claims 47 to 55, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.

58. The nucleic acid analysis apparatus of claim 57, wherein the automated nucleic acid analysis platform is a point-of care assay instrument.

59. The nucleic acid analysis apparatus of claim 58, wherein the point-of-care assay instrument is adapted for pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection.

60. The nucleic acid analysis apparatus of claim 58, wherein the point-of-care assay instrument is adapted for cancer detection.

61. The use of claim 56, or the nucleic acid analysis apparatus of any one of claims 57 to 60, wherein the platform is adapted to perform protein isolation and/or analysis steps.

62. The method of any one of claims 11 to 46, wherein the isolation of protein comprises contacting the sample with the immobilized protein affinity reagent in the aqueous nucleic acid purification buffer that is used for the sequential isolation of DNA and RNA.