Sequential nucleic acid isolation

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 are kits comprising the purification buffer and solid support, use of the kits in an automated nucleic acid analysis platform, and a nucleic acid analysis apparatus comprising an automated nucleic acid analysis platform and the above kits.

Description

[0001] This invention relates to a method for sequentially isolating DNA and RNA from a sample, and related kits and apparatus. The method involves binding DNA and RNA to different solid supports. Upstream processes, such as lysis (e.g., releasing nucleic acids from a biological sample into solution), and / or downstream processes, such as amplification, detection, analysis, etc., are also provided. Background Technology

[0002] When extracting rare targets (such as tumor-derived analytes (e.g., circulating tumor cells (CTCs)), tumor-derived proteins, ctDNA, and ctRNA) for multi-omics-based analyses, capturing as many available analytes as possible in the sample matrix is ​​crucial. Because current multi-omics analyses require different readout methods depending on the analyte (e.g., mass spectrometry or immunoassay for proteomics, NGS for genomics and epigenomics, and transcriptomics), it is necessary to extract protein, DNA, and RNA targets separately to enable different analytical workflows. Currently, protein and nucleic acid extraction require different extraction chemistry and solid phases. However, there is significant overlap between DNA and RNA extraction, making efficient separation of the two particularly challenging. This challenge is currently addressed in part by altering the cleavage-binding buffer chemistry between DNA and RNA extractions. Unfortunately, specific separation of DNA-only targets versus RNA-only targets is challenging due to the following factors: despite alterations to the chemical composition between RNA and DNA extractions, residual levels of the corresponding targets remain high, resulting in the loss of valuable RNA or DNA targets; changes to buffer composition require the addition of extra buffer components to an already complex workflow; and the precipitation of predominantly nucleic acid-based alcohol- or polyol-based materials, which makes the chemicals either flammable (i.e., alcohol-based—and therefore unsuitable for POC kits) or highly viscous (polyol-based, thus challenging to automate on liquid handling systems). Changing the buffer composition also dilutes the original sample and, consequently, even further, the molecules to be separated. Separating proteins before separating nucleic acids enhances this effect.

[0003] For Qiagen's AllPrep® DNA / RNA / Protein, the lysate is passed through an AllPrep DNA centrifuge column, which, in combination with a high-salt buffer, allows for selective binding of genomic DNA. The column is then washed, eluting the bound DNA. Ethanol is then added to the flow-through from the centrifuge column to provide appropriate binding conditions for RNA. The sample is then applied to an RNeasy centrifuge column, where total RNA binds to the membrane. Therefore, the Qiagen AllPrep® DNA / RNA / Protein kit requires modifications to both the lysis binding buffer chemistry and the solid phase to facilitate sequential DNA and RNA extraction.

[0004] For Cytiva's triplePrep kit, the dissociation salt in the type 15 lysis buffer promotes DNA binding to the silica membrane. The DNA-bound silica membrane is then washed, and the DNA is eluted. Acetone is then added to the flow-through buffer, which, in the presence of the dissociation salt in the type 15 lysis buffer, promotes the binding of total RNA to the silica membrane. Therefore, Cytiva's triplePrep kit requires modification of the lysis binding buffer chemistry to facilitate sequential DNA and RNA extraction.

[0005] Therefore, there is a need for improved methods to efficiently and sequentially separate DNA, RNA, and optionally proteins from the same sample. This is particularly relevant when sample volumes are only moderate and / or the concentrations of one or more target molecules within the sample are low, thus strategies involving sample splitting and / or dilution may result in the amount of separated molecules being below the detection limit. Furthermore, such improved methods could enable efficient high-throughput automation to POC-based sample processing automation, providing multi-omics readouts for early cancer diagnosis, early neurological disease diagnosis, precision medicine, and treatment response monitoring. Summary of the Invention

[0006] A first aspect of the present invention provides a method for sequentially separating DNA and RNA from a sample, the method comprising steps a) to d):

[0007] a) In the presence of an aqueous nucleic acid purification buffer, the sample is contacted with a solid support containing surface silanol groups to provide a solid support for DNA binding;

[0008] b) Remove the solid support containing the DNA from the sample;

[0009] c) In the presence of this aqueous nucleic acid purification buffer, contact the sample with a solid support containing surface carboxyl groups to provide a solid support for RNA binding; and

[0010] d) Remove the solid support for RNA binding from the fluid sample.

[0011] The aqueous nucleic acid purification buffer contains a polar aprotic solvent.

[0012] The polar aprotic solvent can be present in an amount of at least about 2% by weight. The polar aprotic solvent can be present in an amount of no more than about 80% by weight. For example, the polar aprotic solvent can be present in an amount between about 4% by weight and about 75% by weight.

[0013] Polar aprotic solvents can have boiling points exceeding 100°C at 1 atm. For example, polar aprotic solvents can have boiling points exceeding 150°C at 1 atm.

[0014] Polar aprotic solvents may contain 2, 3, or 4 heteroatoms selected from O and N. For example, a polar aprotic solvent may contain 2 or 3 heteroatoms selected from O and N. A polar aprotic solvent may contain 2 or 3 O atoms. A polar aprotic solvent may contain 1 or 2 O atoms and 1 or 2 N atoms. A polar aprotic solvent may contain 2 O atoms. A polar aprotic solvent may contain 3 O atoms. A polar aprotic solvent may contain 1 O atom and 1 or 2 N atoms. O atoms may contain =O and / or -O-.

[0015] Polar aprotic solvents can contain 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, a polar aprotic solvent can contain 5, 6, 7, or 8 carbon atoms; for instance, a polar aprotic solvent can contain 6 or 8 carbon atoms. A polar aprotic solvent can contain 5 carbon atoms. A polar aprotic solvent can contain 6 carbon atoms. A polar aprotic solvent can contain 7 carbon atoms. A polar aprotic solvent can contain 8 carbon atoms.

[0016] At 20°C and 1 atm, polar aprotic solvents can have a viscosity of less than 50 cP. For example, at 20°C and 1 atm, polar aprotic solvents can have a viscosity of less than 40 cP or 30 cP, and for example, at 20°C and 1 atm, polar aprotic solvents can have a viscosity of less than 25 cP.

[0017] Polar aprotic solvents may have a flash point of at least 50°C.

[0018] Polar aprotic solvents can be liquid at 0 °C and 1 atm.

[0019] Polar aprotic solvents can be non-flammable, non-sticky, EHS-friendly, and / or bio-renewable.

[0020] Polar aprotic solvents can be environmentally, health, and safety (EHS) friendly solvents.

[0021] The polar aprotic solvent may be selected from one or more of the following: dihydro-L-glucanone, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, and N-formylmorpholine. For example, the polar aprotic solvent may be selected from one or more of the following: dihydro-L-glucanone, N-butylpyrrolidone-2-one, and dipropylene glycol dimethyl ether. In a preferred embodiment, the polar aprotic solvent is N-butylpyrrolidone-2-one.

[0022] The purification buffer may further contain one or more of the following: a dissociating agent, a surfactant, a buffer, an enzyme, an inorganic salt, an antifoaming agent, or a combination thereof. For example, the purification buffer may contain two or more of the following: a dissociating agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent; or, for example, the purification buffer may contain three or more of the following: a dissociating agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent.

[0023] The purification buffer may contain a moderate amount (e.g., less than about 20%, less than about 10%, or less than about 5%) of alcohol or polyol. A purification buffer having no more than a moderate amount (e.g., less than about 5%, or less than about 2%) of alcohol or polyol is preferred.

[0024] The purification buffer may contain a moderate amount (e.g., less than about 20%, less than about 10%, or less than about 5%) of polyethylene glycol (PEG), TEG, or LPA). A purification buffer containing no more than a moderate amount (e.g., less than about 5%, or less than about 2%) of PEG, TEG, or LPA is preferred.

[0025] Purification buffers can be binding buffers and / or washing buffers. For example, purification buffers can be both binding and washing buffers. Purification buffers can be binding buffers. Purification buffers can be washing buffers. Binding conditions for precipitating nucleic acids onto a solid support may be more stringent than washing conditions that require preventing nucleic acids from eluting from the solid support.

[0026] Therefore, in some embodiments, the wash buffer may represent an aqueous dilution of the binding buffer. The aqueous dilution may also contain other reagents that may not be present in the binding buffer, such as buffers, or buffers and inorganic salts. The wash buffer may contain 1.1 to 5 times the aqueous dilution of the binding buffer; for example, the wash buffer may contain 1.5 to 4 times the dilution of the binding buffer.

[0027] In an embodiment, the workflow or kit may include both the binding buffer and the washing buffer of the present invention, wherein the binding buffer and the washing buffer have different compositions, i.e., the washing buffer is neither the same as the binding buffer nor an aqueous dilution of the binding buffer.

[0028] In these examples, the purification buffer does not contain ethanol, isopropanol, or 2-methyl-1,3-propanediol. In these examples, the purification buffer does not contain dimethyl sulfoxide (DMSO). In these examples, the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, or DMSO. In these examples, the purification buffer does not contain alcohols (including any glycols or polyols). In these examples, the purification buffer does not contain alcohols (including any glycols or polyols) or DMSO. In these examples, the purification buffer does not contain salts or dissociation agents.

[0029] When the sample is not a liquid sample (i.e., a solid sample), the method may first involve suspending or dissolving the solid sample in a suitable buffer (e.g., an aqueous nucleic acid purification buffer).

[0030] This method can be a method for sequentially separating proteins, DNA, and RNA from a sample, and the method further includes separating proteins prior to step a).

[0031] Protein separation can be targeted or non-targeted. One or more targeted protein separations can be followed by one or more non-targeted protein separations.

[0032] Isolation of proteins from a sample may include targeted affinity protein separation, such as immunoprecipitation. Affinity protein separation is conveniently performed using a solid phase with suitable surface properties via appropriate methods. The appropriate method will depend on the type of target protein. In one embodiment, a specific protein may be separated using a solid support having suitable binding partners / ligands attached to its surface. This thus applies well-known principles of protein affinity separation as widely described in the prior art. Any such standard and well-known method may be used or adapted to this invention.

[0033] Targeted affinity protein separation may include: contacting a sample with a specific protein affinity reagent immobilized to a solid support to provide a protein-binding solid support; and removing the protein-binding solid support from a fluid sample. This method may include contacting a sample with an immobilized protein affinity reagent in an aqueous nucleic acid purification buffer for subsequent sequential separation of DNA and RNA. The protein affinity reagent may be any molecule with affinity for the target protein, such as antigens, antibodies, lectins, carbohydrates, metal ions, engineered protein scaffolds, substrates, and / or inhibitors of the target protein. Affinity reagents are well known to those skilled in the art and are commercially available or may be manufactured separately, for example, using an antibody-conjugation kit that allows the user to covalently conjugate antibodies or antigens to a solid support, such as Dynabeads. TM Magnetic beads for use in immunoprecipitation workflows.

[0034] Targeted affinity protein separation (e.g., immunoprecipitation) typically requires diluting the sample in an appropriate phosphate buffer (such as PBS or PBST buffer). Targeted affinity protein separation can be performed sequentially or simultaneously, one or more times (e.g., two, three, four, etc.). When performing more than one targeted affinity separation, using beads of different sizes or with different labels can allow for the differentiation of different separated proteins (if desired).

[0035] Alternatively, or subsequently, solid phases with more general surface binding properties can be selected, such as solid phases with surface chemistry properties that influence typical chromatographic interactions (e.g., ion exchange, including both anion and cation exchange), reversed-phase interactions, or hydrophobic interactions. Such surfaces can be conveniently provided on magnetic beads. The use of these more general surfaces conveniently allows for the fractionation of proteins into subsets based on their structure and properties (charge, hydrophobicity, etc.) in the sample. Some of these general protein enrichment methods utilize weak to strong anionic or cationic surface chemistry. Such conventional solid phase surfaces can be prepared using conventional techniques that are standard and well-documented in the field of column chromatography. Therefore, protein separation from a sample may include, for example, contacting the sample with a solid support containing ionizable surface groups, lowering the pH of the sample to ionize the surface groups, thereby allowing the ionized surface groups to selectively bind both DNA and RNA to form a nucleic acid-binding solid support. The nucleic acid-bound solid support can then be removed from the protein, optionally washed (e.g., with washing buffer) and contacted with elution buffer to separate the DNA and RNA from the solid support. In another embodiment, SAX (strong anion exchange) is used, where negatively charged proteins or exosomes / viruses bind to the solid support under physiological or low-salt conditions. The protein can then be eluted from the support using high salt (e.g., 1M NaCl). The solution containing DNA and RNA not bound to the solid support forms the (protein-free) sample used in step a). This sample is typically added to an aqueous nucleic acid purification buffer, or... vice versa .

[0036] The method may further include a lysis step. If necessary, for example, if the nucleic acids and proteins to be separated are not bound within the initial sample (e.g., contained within biological particles such as viral coatings or cell membranes or walls), one or more separate steps can be performed prior to the initial binding step to release the nucleic acid and protein components, for example, by disrupting structural components such as cell walls, to achieve lysis. Procedures for achieving this are well known in the art. Thus, for example, while some cells (e.g., blood cells) can be lysed simply by reagents (such as detergents), other cells (e.g., plant or fungal cells or solid animal tissues) may require more forceful treatment, such as, for example, grinding in liquid nitrogen, heating in the presence of detergents, alkaline lysis in the presence of detergents, or freeze / thaw cycles. In many cases, the procedure for releasing the nucleic acids and proteins is chosen such that the specific nucleic acid and protein species to be separated in the method of the present invention remain sufficiently intact, for example, substantially undegraded, and if an affinity method is used in the optional protein separation step, the lysis conditions do not adversely affect any antibody or antigen or their mutual affinity.

[0037] Typically, any lysis step is performed before or during step a). The lysis step may be performed before proteins are separated from the sample. The lysis step may be performed after proteins are separated from the sample. The lysis step may also be performed between targeted separations of two or more different proteins, or between targeted protein separation and more general protein separation.

[0038] The lysis step may include adding a lysis buffer to the sample. After combining the sample and lysis buffer, additional lysis steps, such as agitation, heating, and / or incubation, may be performed. The lysis buffer may be an aqueous nucleic acid purification buffer. For some samples, the lysis step can be combined with the binding step a) by adding a polar aprotic solvent and a solid support containing surface silanol groups to the lysis buffer, providing a one-step lysis and binding workflow. The polar aprotic solvent and the solid support containing surface silanol groups may be added before combining the sample and lysis buffer, or after combining the sample and lysis buffer and optional additional lysis steps (such as agitation and / or incubation).

[0039] Lysis or lysis / nucleic acid purification buffers may contain high concentrations of dissociation agents (e.g., guanidine salts or urea) and surfactants such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, octyl-β-glucan, octylthioglucan, SDS (sodium dodecyl sulfate), CHAPS, and / or CHAPSO, preferably Tween-20. Lysis or lysis / binding buffers may contain any of the components disclosed herein in connection with the methods, uses, and compositions described herein. Lysis buffers may contain enzymes such as lysozyme, proteolytic enzymes (such as proteinase K), etc.

[0040] The method may further include contacting the sample with a proteolytic enzyme (e.g., proteinase K) prior to step a), provided that the proteolytic enzyme / proteinase K is not added until after (optionally) protein separation. The method may further include contacting the sample with proteinase K and lysis buffer prior to step a), provided that the proteinase K and lysis buffer are not added until after (optionally) protein separation.

[0041] Typically, steps a) and b) are performed before steps c) and d). However, steps c) and d) may also be performed before steps a) and b).

[0042] In contact step a), the different components (such as the sample, a suitable buffer, a solid support containing surface silanol groups, and a polar aprotic solvent) can be added in any possible order. Therefore, the method may include adding a solid support containing surface silanol groups to a sample already mixed with a suitable buffer, and adding a polar aprotic solvent as the final component. For example, the suitable buffer may be mixed with the polar aprotic solvent first, then the sample is added, and finally the solid support is added. An aqueous nucleic acid purification buffer containing a polar aprotic solvent and a solid support containing surface silanol groups may be added to the sample sequentially or simultaneously; for example, the aqueous nucleic acid purification buffer may contain a solid support containing surface silanol groups dispersed therein. An excess of a solid support containing surface silanol groups may be added to the sample.

[0043] Typically, this method will include adding a solid support containing surface carboxyl groups to the sample after step b).

[0044] Step b) may further include optional step b2): washing the DNA-bound solid support with a wash buffer. Step b2) may include washing the DNA-bound solid support at least once. Step b2) may include washing the DNA-bound solid support at least twice, for example at least three times, each time using fresh wash buffer. The wash buffer may contain a purification buffer. In some instances, the wash buffer may be the same as the purification buffer (or an aqueous dilution of the purification buffer). In other instances, the purification buffer and the wash buffer have different compositions, i.e., the wash buffer is neither the same as the binding buffer nor an aqueous dilution of the binding buffer.

[0045] The binding conditions for precipitating nucleic acids onto a solid support may be more stringent than the washing conditions required to prevent the nucleic acids from eluting from the solid support. Therefore, a wash buffer can represent an aqueous dilution of the purification buffer used for binding. For example, a wash buffer may contain 1.1 to 5 times the concentration of the purification buffer; or, for example, a wash buffer may contain 1.5 to 4 times the concentration of the purification buffer.

[0046] Step b2) may include washing the DNA-bound solid support with wash buffer 1. Wash buffer 1 may contain a nucleic acid purification buffer. Wash buffer 1 may contain at least one polar aprotic solvent (e.g., a polar aprotic solvent present in aqueous nucleic acid purification buffers, such as dihydro-L-glucosamine, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, or N-formylmorpholine; for example, N-butylpyrrolidone-2-one). Wash buffer 1 may contain about 25% to about 75% by volume of a polar aprotic solvent, such as dihydro-L-glucosamine, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, or N-formylmorpholine; for example, N-butylpyrrolidone-2-one. Washing buffer 1 may contain about 35 vol% to about 65 vol% of a polar aprotic solvent, such as dihydro-L-glucosamine, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, or N-formylmorpholine; for example, N-butylpyrrolidone-2-one. Washing buffer 1 may contain about 45 vol% to about 55 vol% of a polar aprotic solvent, such as dihydro-L-glucosamine, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, or N-formylmorpholine; for example, N-butylpyrrolidone-2-one.

[0047] Wash buffer 1 may contain a polar organic solvent (different from or supplemented with the polar aprotic solvent present in the aqueous nucleic acid purification buffer). The polar organic solvent may be an alcohol, such as C1-C6 alcohols, polyols, or oligoethylene glycol. The polar organic solvent may be a C1-C6 alcohol. The polar organic solvent may be isopropanol.

[0048] Wash buffer 1 may contain about 25 vol% to about 75 vol% of an organic solvent (e.g., isopropanol). Wash buffer 1 may contain about 35 vol% to about 65 vol% of an organic solvent (e.g., isopropanol). Wash buffer 1 may contain about 45 vol% to about 55 vol% of an organic solvent (e.g., isopropanol).

[0049] Step b2) may include washing the DNA-bound solid support with wash buffer 2. Wash buffer 2 may not contain nucleic acid purification buffer. Wash buffer 2 may contain an organic solvent (e.g., a polar organic solvent) and optionally water. Wash buffer 2 may contain more than or equal to about 25% by volume of organic solvent. Wash buffer 2 may contain more than or equal to about 30% by volume, for example, about 35% by volume of organic solvent. Wash buffer 2 may contain less than or equal to about 90% by volume of organic solvent. Wash buffer 2 may contain less than or equal to about 80% by volume of organic solvent. At least some (e.g., all) of the remaining volume of wash buffer 2 may be water.

[0050] The organic solvent may be an alcohol, such as C1-C6 alcohols, glycols (e.g., methylpropanediol), polyols, or oligoethylene glycols. The organic solvent may be a C1-C6 alcohol. The alcohol may be ethanol or isopropanol. Wash buffer 2 may contain about 50% to about 90% by volume of an alcohol (e.g., ethanol). Wash buffer 2 may contain about 60% to about 80% by volume of an alcohol (e.g., ethanol). The organic solvent may be a glycol ether, such as a C2-C6 alcohol. 10 Glycol ether. The glycol ether may be 2-butoxyethanol. Wash buffer 2 may contain about 25% to about 75% by volume of glycol ether (e.g., 2-butoxyethanol). Wash buffer 2 may contain about 35% to about 65% by volume of glycol ether (e.g., 2-butoxyethanol).

[0051] Wash buffer 2 may contain less than about 50% by volume of N-butylpyrrolidone. Wash buffer 2 may contain less than about 25% by volume of N-butylpyrrolidone. Wash buffer 2 may contain less than about 5% by volume of N-butylpyrrolidone. Wash buffer 2 may not contain N-butylpyrrolidone.

[0052] Step b2) may include washing the DNA-bound solid support with at least two different washing buffers. For example, step b2) may include washing the DNA-bound solid with washing buffer 1 and washing buffer 2, wherein washing buffer 1 and washing buffer 2 are different. Step b2) may include washing the DNA-bound solid support with washing buffer 1 before washing the DNA-bound solid support with washing buffer 2.

[0053] Step b) may further include, optionally, step b3): contacting the DNA-bound solid support with an elution buffer to separate the DNA from the solid support. The elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof. Conventional elution buffers may be used, such as aqueous Tris buffers with a pH greater than 7, such as 10 Mm Tris-HCl buffer with a pH of 8.0.

[0054] Step d) may further include optional step d2): washing the RNA-bound solid support with a wash buffer. Step d2) may include washing the RNA-bound solid support at least once. Step d2) may include washing the RNA-bound solid support at least twice, for example at least three times, each time using fresh wash buffer. The wash buffer may be or may contain a purification buffer. In some instances, the wash buffer may be the same as the purification buffer (or an aqueous dilution of the purification buffer). In other instances, the purification buffer and the wash buffer have different compositions, i.e., the wash buffer is neither the same as the binding buffer nor an aqueous dilution of the binding buffer.

[0055] The binding conditions for precipitating nucleic acids onto a solid support may be more stringent than the washing conditions required to prevent the nucleic acids from eluting from the solid support. Therefore, a wash buffer can represent an aqueous dilution of the purification buffer used for binding. For example, a wash buffer may contain 1.1 to 5 times the concentration of the purification buffer; or, for example, a wash buffer may contain 1.5 to 4 times the concentration of the purification buffer.

[0056] Step d2) may include washing the RNA-bound solid support with at least two different wash buffers. For example, step d2) may include washing the RNA-bound solid with wash buffer 1 and wash buffer 2, wherein wash buffer 1 and wash buffer 2 are different. Step d2) may include washing the RNA-bound solid support with wash buffer 1 before washing it with wash buffer 2. Wash buffer 1 and wash buffer 2 may be as defined above with respect to step b2).

[0057] Step d) may further include, optionally, step d3): contacting the RNA-bound solid support with an elution buffer to separate the RNA from its solid support. The elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof. Conventional elution buffers may be used, such as, for example, an aqueous Tris buffer with a pH greater than 7, such as a 10 mM Tris-HCl buffer with a pH of 8.0.

[0058] In embodiments, the method includes: contacting a 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 include contacting the sample with the immobilized protein affinity reagent in an aqueous nucleic acid purification buffer for subsequent sequential separation of DNA and RNA. In these embodiments, the method may further include washing the protein-bound solid support with a wash buffer. For example, the method may include washing the protein-bound solid support at least once. The method may include washing the protein-bound solid support at least twice, for example at least three times, each time using fresh wash buffer. The wash buffer may be or may contain physiological buffers, PBS, HEPES, Tris, NH4OAc, etc., with or without salts (e.g., KOAc, NaCl, MgCl2, KCl), with or without ionic or nonionic, 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 and the subsequent use of the separated target protein.

[0059] The method may further include contacting the protein-binding solid support with an elution buffer to separate the protein from the solid support. The elution buffer may include an aqueous salt solution, such as saline, or a solution containing glycine-HCl with a pH of about 2.5-3.0.

[0060] The isolated proteins, DNA, and / or RNA may undergo one or more additional processes, such as identification and / or quantification. These additional downstream processes may be selected from, but are not limited to, microarray analysis, detection, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, epigenetic analysis, sequencing, amplification, transfection, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulations, and genome editing.

[0061] Amplification can include PCR, qPCR, digital PCR (dPCR), reverse transcription, in vitro transcription, or isothermal amplification. Isothermal amplification can include 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 crossdisplacement amplification (MCDA), signal-mediated RNA amplification (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 its references).

[0062] Sequencing can include next-generation sequencing. "Next-generation sequencing" and "high-throughput sequencing" are sequencing technologies that parallelize the sequencing process, producing a large number of sequences at once. Next-generation sequencing methods can include single-molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 LifeSciences), sequencing by ligation (e.g., SOLiD sequencing from Thermo Fisher Scientific's AppliedBiosystems), sequencing by synthesis, and reversible terminator sequencing (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 Biosciences), hybridization sequencing, and nucleic acid imaging techniques (such as transmission electron microscopy).

[0063] The isolated DNA may be further subjected to one or more additional processes, selected from, but not limited to, microarray analysis, qPCR, dPCR, and next-generation sequencing.

[0064] The isolated RNA may further undergo one or more additional processes, selected from, but not limited to, reverse transcription, microarray analysis, qPCR, dPCR, and next-generation sequencing.

[0065] The process of this invention can also be used for the extraction and subsequent analysis of fragmented DNA.

[0066] This method may further include point-of-care (POC) testing. POC testing may include, for example, testing for clinical biomarkers or drugs, pathogen detection and / or biological warfare agent detection and / or genetic disease detection. Pathogen detection or biological warfare agent detection may include detection of nucleic acids and / or proteins of viruses, bacteria, single-celled fungi, or protozoa. POC testing may be a single test or multiplex detection. Testing for clinical biomarkers or drugs and genetic disease detection may include prenatal testing.

[0067] Any isolated protein may be further subjected to one or more additional processes. Following targeted affinity separation, the protein can be quantified. Following non-targeted separation, the protein can be further separated, identified, and / or quantified using methods well-known in the art. One or more additional processes may be selected from, for example, protein microarray analysis, mass spectrometry, light scattering, immunoassays (such as ELISA or Western blotting), protein sequencing, enzyme activity assays, and / or other methods known in the art.

[0068] Solid supports containing surface silanol groups may include one or more of the following: particles, resins, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. Solid supports containing surface silanol groups may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports containing surface silanol groups may be components of robotic liquid handling platforms.

[0069] Solid supports containing surface carboxyl groups may include one or more of the following: particles, resins, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. Solid supports containing surface carboxyl groups may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports containing surface carboxyl groups may be components of robotic liquid handling platforms.

[0070] Solid supports to which protein affinity reagents (e.g., antigens, ligands, antibodies, peptides, aptamers, lectins, carbohydrates, metal ions, engineered protein scaffolds, substrates, and / or inhibitors or metabolites of target proteins) are immobilized may include one or more of the following: particles, resins, beads, filters, boxes, columns, arrays, membranes, chips, disks, or slides. Solid supports to which protein affinity reagents are immobilized may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports to which protein affinity reagents are immobilized may be components of robotic liquid handling platforms.

[0071] Solid supports containing ionizable surface groups may include one or more of the following: particles, resins, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. Solid supports containing ionizable surface groups may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports containing ionizable surface groups may be components of robotic liquid handling platforms.

[0072] DNA can be one or more of the following: synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g., dsDNA or ssDNA), cDNA, cfDNA, gDNA, or ctDNA. DNA can be ctDNA.

[0073] For example, RNA can be one or more of the following: mRNA (e.g., isolated from biological samples or...) in vitro Transcribed RNA, siRNA, microRNA, tRNA, cfRNA, rRNA, viral RNA (e.g., dsRNA or ssRNA), snRNA, or ctRNA. RNA can be ctRNA.

[0074] sample

[0075] Samples may include or may be pretreated or untreated biological samples, clinical or environmental samples, or enzymatic reaction mixtures. Samples may be or may include pretreated or untreated biological samples. Biological samples may be in physiological buffers or transfer media. Biological samples may be harvested or biopsied samples or cultured samples. Samples may be or may contain environmental samples. Samples may be or may contain enzymatic reaction mixtures.

[0076] Biological samples can be any suitable biological sample. Exemplary biological samples may include, but are not limited to, one or more of the following: blood, bloodstains, umbilical cord blood, blood components (e.g., concentrated platelets), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, and erythrocyte sedimentation rate (ESR) buffy layer. Leukocytes, serum, plasma, saliva, saliva stains, buccal cells, buccal swabs, semen, semen stains, urine, feces, fecal stains, cigarette butts, chewing gum, formalin-fixed paraffin-embedded (FFPE) samples, biopsy (e.g., tumor biopsy) samples, bone marrow or other tissue samples, plant samples, cell lysates, bacterial or yeast cultures, sputum, tears, throat swabs, oral irrigant, nasopharyngeal swabs, nasopharyngeal aspirates, exhaled fluid, nasal swabs, nasal irrigant, mucus, bronchial aspirates, bronchoalveolar lavage fluid, pleural fluid, tracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, cervical swabs, vitreous fluid, amniotic fluid, breast milk, exosomes, circulating tumor cells, tissue lysates, bacterial lysates, yeast lysates, and plant lysates. Biological samples may include exosomes. Biological samples may be fluid biological samples. In some cases, biological samples can be clinical samples. In other cases, samples can be cell-free samples.

[0077] Environmental samples may include water samples (such as wastewater samples, swimming pool water samples, seawater samples), soil samples, sediment samples, surface swabs, air-derived samples (such as air filter residues), cosmetics, food ingredients or food samples, or combinations thereof.

[0078] The enzyme reaction mixture can be any such mixture that may contain a nucleic acid sequence. For example, the enzyme reaction mixture may include an 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 enzyme digestion reaction mixture, a nucleic acid assembly reaction mixture, or a barcoding reaction mixture.

[0079] The method of this invention advantageously allows for the sequential extraction of RNA and DNA present in very low quantities in large sample volumes (e.g., cfDNA or cfRNA, ctDNA or ctRNA in plasma) or from small samples (e.g., single or small-volume detection samples, as is typically the case with CTC). Thus, samples can contain DNA and / or RNA at levels from about 1 picogram of nucleic acid / mL (or even lower) to about 1 microgram of nucleic acid / mL (or even higher). qPCR or RTqPCR can be used for the detection and quantification of DNA / RNA in the sub-picogram range. In the picogram to nanogram range, detection and quantification can be performed using fluorescent nucleic acid intercalation dyes.

[0080] The specific DNA and RNA to be isolated will vary depending on the sample. For example, the sample may include CTC, the DNA may include gDNA, and the RNA may include total RNA. The sample may include exosomes, the DNA may include cfDNA, and the RNA may include one or more of the following: miRNA, mRNA, and snRNA.

[0081] A second aspect of the present invention provides a kit comprising:

[0082] A first solid support comprising surface silanol groups;

[0083] A second solid support, the second solid support comprising surface carboxyl groups; and

[0084] An aqueous nucleic acid purification buffer containing a polar aprotic solvent.

[0085] The kit may additionally include components for the separation of one or more targeted proteins and / or non-targeted proteins, such as a third solid support containing immobilized protein affinity reagents, such as, for example, a toluenesulfonyl activated surface for binding antibodies against the target protein to be separated, or a solid support having a surface suitable for binding to the target protein, protein subgroup, or all proteins in the sample.

[0086] The first solid support and the second solid support may be as defined in the first aspect above.

[0087] Aqueous nucleic acid purification buffers and polar aprotic solvents are as defined in the first aspect above.

[0088] The kit may contain one or more of the following: lysis or lysis / binding buffer, washing buffer, and elution buffer. The lysis buffer and / or washing buffer may contain a polar aprotic solvent of an aqueous nucleic acid purification buffer. The kit may contain two different washing buffers (washing buffer 1 and washing buffer 2). Washing buffer 1 may be as defined above with respect to the first aspect of the invention. Washing buffer 2 may be as defined above with respect to the first aspect of the invention.

[0089] The third aspect of the present invention provides the use of the kit of the second aspect of the present invention in a nucleic acid analysis platform.

[0090] A fourth aspect of the present invention provides a nucleic acid analysis device, comprising:

[0091] Automated nucleic acid analysis platform; and

[0092] The kit according to any of the second aspects of the present invention,

[0093] The automated nucleic acid analysis platform includes a section configured to accommodate solid support.

[0094] Automated nucleic acid analysis platforms can be point-of-care testing instruments. Point-of-care testing instruments can be used for pathogen detection and / or biological warfare agent detection, clinical biomarkers or drug and / or genetic disease detection. Pathogen detection or biological warfare agent detection may include nucleic acid detection of viruses, bacteria, single-celled fungi, or protozoa. In a preferred embodiment, point-of-care testing instruments are suitable for cancer detection. Testing for clinical biomarkers, pathogens, or drugs, as well as genetic disease detection, may include prenatal testing.

[0095] This invention has a wide range of applications in laboratory research, human and veterinary medicine, public health and sanitation, forensic medicine, anthropological research, environmental monitoring, and industry. These applications include, but are not limited to, bacterial and viral detection and typing, antimicrobial resistance screening, viral load determination, genotyping, infection control and pathogen screening (for example, blood, tissue, food, cosmetics, water, soil, and air), pharmacogenomics, detection of cell-free DNA in plasma, white blood cell counting, and other areas of interest in the preparation and analysis of DNA from biological samples. Attached Figure Description

[0096] Embodiments of the present invention will now be further described with reference to the accompanying drawings, wherein:

[0097] Figure 1 provides an exemplary schematic diagram illustrating a general process for sequentially separating proteins, DNA, and RNA from a sample.

[0098] Figure 2 provides an exemplary schematic diagram illustrating a more detailed process for sequentially separating proteins, DNA, and RNA from liquid biopsy samples.

[0099] Figure 3 is a bar graph showing the recovery of nucleic acids from silane and carboxylic acid functionalized stationary phases using the aprotic solvent N-butylpyrrolidone-2-one (TamiSolve™) in binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. Detailed Implementation

[0100] Throughout the description and claims, the terms “comprise” and “contain” and their variations mean “including, but not limited to,” and are not intended to exclude (and do not exclude) other parts, additives, components, integers, or steps. Throughout the description and claims, the singular form includes the plural form unless the context otherwise requires. In particular, where the indefinite article is used, the description should be understood to consider both the plural and singular forms unless the context otherwise requires.

[0101] Features, integers, properties, compounds, chemical parts, or groups described in connection with specific aspects, embodiments, or examples of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed can be combined in any combination, except for at least some mutually exclusive combinations of such features and / or steps. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination of steps of any method or process so disclosed.

[0102] Readers should note all documents and files associated with this application that were submitted concurrently with or prior to this specification and made publicly available together with this specification, and the contents of all such documents and files are incorporated herein by reference.

[0103] To avoid any doubt, it is hereby declared that the information disclosed above under the heading "Background Art" is relevant to this invention and should be understood as part of the disclosure of this invention.

[0104] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0105] definition

[0106] The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in implementing it.

[0107] Unless otherwise stated, the term "nucleic acid" means a polynucleotide molecule composed of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotide residues capable of participating in Watson-Crick-type or similar base pair interactions (i.e., the formation of a "hybrid" or "double strand"). Thus, nucleic acids can be DNA or RNA or any modification thereof, including conformationally restricted or nucleobase analogs of oligomers, such as "locked nucleic acids" (LNAs) or "peptide nucleic acids" (PNAs), or other derivatives containing a non-nucleotide backbone. Nucleic acids can be naturally occurring molecules, i.e., DNA or RNA, but also include DNA / RNA hybrids (where DNA is in separate strands or in the same strand), where the 3' position of the pentose sugar of one nucleotide is linked to the 5' position of the pentose sugar of the next nucleotide via a phosphodiester bond. Nucleic acids used in the various embodiments may include nucleotides or combinations thereof in the form of chemically, enzymatically, or metabolically modified forms, 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).

[0108] Unless the context otherwise requires, the term "purification buffer" means a buffer that can be used to precipitate nucleic acids from solution onto a solid support and / or a buffer that can be used to wash nucleic acids bound to a solid support. Therefore, an exemplary purification buffer can also be considered as a binding buffer or a washing buffer.

[0109] Unless the context otherwise requires, the term "binding buffer" refers to a buffer solution that can be used to precipitate nucleic acids from solution onto a solid support. Nucleic acids are typically solvated in aqueous solutions, so typical binding buffers are miscible in aqueous solutions; for example, binding buffers can be provided in the form of aqueous buffers. An exemplary binding buffer of the present invention is an aqueous buffer containing a polar aprotic solvent.

[0110] Unless the context otherwise requires, the term "wash buffer" refers to a buffer solution that can be used to wash nucleic acids bound to a solid support. Given that binding buffers can precipitate nucleic acids from solution, they can also be used as wash buffers, as they should minimize the loss of nucleic acids from the solid support during washing. Binding conditions that precipitate nucleic acids onto a solid support may be more stringent than washing conditions that require preventing the elution of nucleic acids from the solid support. Therefore, wash buffer can represent an aqueous dilution of the binding buffer, such as a 1.1 to 5-fold dilution of the corresponding binding buffer.

[0111] Unless the context otherwise requires, the term "elution buffer" means a buffer that can be used to elute (remove) a substance from a solid support (containing, for example, nucleic acids or proteins). The elution buffer used will depend on the substance to be eluted and the solid support. For example, a nucleic acid elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof. On the other hand, a protein elution buffer may include: an aqueous salt solution (such as saline), a solution containing glycine-HCl at a pH of about 2.5-3.0, a solution containing NH4OH, and / or a buffer suitable for proteolytic digestion (e.g., trypsin digestion) of the eluted protein for, for example, subsequent mass spectrometry analysis.

[0112] Targeted protein separation is the separation of a specific protein or class of proteins. An example of targeted protein separation is immunoprecipitation, in which an antibody with a specific affinity for a protein of interest immobilized to a solid support (e.g., beads) is contacted with the sample, thereby causing the protein to bind to the support (e.g., beads).

[0113] Non-targeted protein separation does not target a specific type of protein and aims to isolate a specific subgroup of proteins (e.g., all lipophilic proteins) or all proteins from a sample. An example of non-targeted protein separation is contacting the sample with a solid support containing silica, wherein the surface has silanol groups or alkyl chains of a certain length (e.g., C4, C8, or C4). 18 This can be combined with other methods. In this example, the solid support may be the stationary phase of a column (e.g., a reversed-phase column). The terms "protein isolation" and "protein separation" are used synonymously.

[0114] Unless otherwise stated, the terms "solid support" or "solid phase" mean a material that is substantially insoluble in a selected solvent system (e.g., containing an aqueous buffer) or that can be readily separated (e.g., by precipitation) from a selected solvent system in which it is soluble. In this disclosure, solid supports that are substantially insoluble in a selected solvent system (e.g., containing an aqueous buffer) are preferred. Such solid supports are not limited to a particular type of support, and a wide variety of such solid supports are available and known to those skilled in the art. Exemplary solid supports include, but are not limited to, solid and semi-solid matrices (such as aerogels and hydrogels), resins, particles, beads (including magnetic beads, such as coated magnetic beads), biochips (including film-coated biochips), microfluidic chips, silicon chips, multiwell plates (also known as microtiter plates or microplates), membranes, conductive and non-conductive metals, glass (including microscope slides), and magnetic supports. When the solid support comprises beads, monodisperse (magnetic or nonmagnetic) beads are preferred because monodisperse beads provide more consistent performance in assays. The solid support may include magnetic beads. Solid supports that can be used to practice the present invention have a hydrophilic surface that allows nucleic acids to bind, for example, through noncovalent interactions. The hydrophilic surface of the solid support may include a charged surface in the pH range of about 6 to about 8.

[0115] The term "monodispersive" means that for a plurality of particles or beads (e.g., at least 100, more preferably at least 1000), the coefficient of variation (CV) or percentage of polydispersity (%) of the particle or bead diameter is less than 20%, for example less than 15%, typically less than 10%, and optionally less than 8%, for example less than 5%. The term "monodispersive" is used herein to characterize a population of particles or beads having low heterogeneity and a uniform size distribution. The size distribution of the particles or beads can be defined by the percentage CV (coefficient of variation), which can be determined on a CPS disc centrifuge, as described, for example, in the Analytical Methods section of WO2017211913A1, which is incorporated herein by reference. CV is defined as 100 multiplied by (standard deviation) divided by the mean, where the "mean" is the average particle or bead diameter of 10, and the standard deviation is the standard deviation of the particle size. The CV for a plurality of particles can, for example, be in the range of 50% to 100%. For example, in a monodisperse group of particles or beads, more than 90%, preferably more than 95%, of the particles or beads may be within ±5% of their average diameter.

[0116] Unless otherwise stated, the term "surface" in relation to a solid support means the solvent-accessible portion of the solid support. This includes any outer surface of the solid support and the surfaces of the solvent-accessible pores of a porous solid support. The surface of a solid support may contain surface hydroxyl groups. Surface hydroxyl groups may contain a portion of a silanol, carboxyl, or sugar moiety. The surface of a solid support may contain negatively charged groups (such as acidic groups) and / or the surface of a solid support may contain positively charged groups. The surface of a solid support may contain acidic groups. The surface of a solid support may contain carboxyl groups. The surface of a solid support may contain positively charged groups, such as one or more pK groups. a Between approximately 5 and 8 (e.g., pK) a Positively ionizable groups (between approximately 6 and 7). The surface of the solid support may contain polyethyleneimine groups, morpholine groups, alanine groups, or polyhydroxyamine groups (such as Tris, Bis-Tris, etc.). The hydrophilic surface of the solid support may contain biological buffers covalently bound thereto; for example, biological buffers as described in column 5, lines 55 through 6, lines 59 of US 6,914,137 B2, covalently bound as described in column 7, lines 29 through 64 of the same document, which is incorporated herein by reference in its entirety.

[0117] A solid support containing surface silanol groups may be made of silicon dioxide, and may contain silicon dioxide on the surface of the solid support. especially With silanol group For the end.

[0118] Solid supports may include beads, such as monodisperse beads. Beads may be monodisperse and / or magnetic and / or porous. (Optionally, monodisperse) beads may be magnetic and / or porous. Beads may be monodisperse; and magnetic and / or porous. Beads may be magnetic, for example, beads may be both monodisperse and magnetic.

[0119] In some cases, magnetic beads may comprise microparticles or nanoparticles. In some instances, magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application number GB2210796.5 (WO 2024 / 018084 A2), which is incorporated herein by reference, may be used. In some instances, the solid support containing surface silanol groups may be selected from any commercially available solid support containing surface silanol groups suitable for binding nucleic acids. For example, a solid support containing surface silanol groups may be selected from Dynabeads. TM MyOne TMSilanes, SeraSil-Mag 400 or 700 (Cytiva), silanol magnetic microspheres (available from VDO Biotech in Suzhou, China, in various nm or µm sizes), and MagneSil TM Beads (available from Promega).

[0120] In some instances, the solid support containing surface carboxyl groups can be selected from any commercially available solid support containing surface carboxyl groups suitable for binding nucleic acids. Solid supports containing surface carboxyl groups can be selected from Dynabeads. TM MyOne TM Carboxylic acids, Dynabeads™ M-270™ carboxylic acids (both available from Thermo Fisher Scientific), and SeraMagSpeedBeads TM Carboxylate modification (Cytiva), BioMagPlus COOH TM and ProMag 1 COOH TM (Both available from Bangs Laboratories, INC Fishers), 4.4 µm fluorescent ferromagnetic beads or 2.0 µm ferromagnetic beads (both available from Spherotech INC, Lake Forest, Illinois), named WHM-S001. TM 2 µm beads, or named WHM-S002 TM 2 µm beads (both available from Creative Diagnostics, New York City, NY), carboxylated magnetic microspheres (available from Weidu Biotechnology Co., Ltd., Suzhou, China, in various nm or µm sizes), carboxylated Adembeads beads (available in 100 nm, 200 nm, 300 nm, or 500 nm sizes), or carboxylated Masterbeads beads (500 nm) (available from Ademtech, France), BeaverBeads TMMag COOH (available from Beaver Biomedical Engineering Ltd.), LodeStars High Bind Carboxyl beads (available from Agilent), Magnosphere™, MS300 carboxyl, MS 160 carboxyl, or MS160 carboxyl (all available from JSR LifeSciences), PureProteome carboxyl FlexiBind magnetic bead system (available from Sigma-Aldrich in various bead sizes), BioMag TM Carboxyl group, BioMag TM Maxi carboxyl group or BioMag TM Plus carboxyl beads (available from Polysciences), Carboxyl Super Mag or Mono Mag magnetic beads (available from Ocean Nanotech in sizes between 0.1 µm and 4.5 µm), and carboxyl beads available from Weidu Biotechnology Co., Ltd. in various sizes.

[0121] In some instances, the solid support used to immobilize the protein affinity reagent may be any commercially available solid support suitable for protein binding. Solid supports for immobilizing protein affinity reagents may be, for example, Dynabeads™ MyOne™ epoxy resin, Dynabeads™ M-270 epoxy resin 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, DynabeadsMyOne 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 (Multi-Species) Magnetic Beads (all available from Thermo Fisher Scientific).

[0122] Unless otherwise stated, the term "alcohol" means a compound containing a hydroxyl group, such as an alkane or alkene substituted with a hydroxyl group. Exemplary alcohols include C1-C6 alcohols; such as methanol, ethanol, propanol, isopropanol, or butanol. In some embodiments described herein, purification buffers, binding buffers, washing buffers, and aqueous media containing aprotic solvents may not contain alcohols.

[0123] Unless otherwise stated, the term "ethylene glycol ether" refers to a compound containing both a hydroxyl group and an ether group. Exemplary ethylene glycol ethers include, but are not limited to, C2-C2 compounds. 10 Ethylene glycol ethers, wherein the ethylene glycol ether may be an alkane or alkene substituted with a hydroxyl group and interrupted by one, two, or three ether bonds, if chemically possible. Exemplary ethylene 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.

[0124] Unless otherwise stated, the term "polyol" means a compound containing a plurality of (e.g., 2) hydroxyl groups. Exemplary polyols include, but are not limited to, C2-C2 groups. 10 Polyols, wherein the polyol may be an alkane or olefin substituted with at least two hydroxyl groups and optionally interrupted by one, two, or three ether bonds. Specific examples of polyols include 2-methyl-1,3-propanediol, tripropylene glycol, and butanediol. In some embodiments described herein, purification buffers, binding buffers, washing buffers, and aqueous media containing aprotic solvents may not contain polyols.

[0125] The term "magnetism" refers to a response to a magnetic field. For example, magnetic beads respond to a magnetic field. Magnetic materials (such as magnetic beads) can be paramagnetic or superparamagnetic. When a magnetic material is paramagnetic, the magnetism turns off when the magnetic field is removed. When a magnetic material is superparamagnetic, it becomes saturated under relatively low magnetic fields, and the magnetism turns off very rapidly / instantaneously when the magnetic field is removed. Some magnetic materials (such as iron oxide) form superparamagnetic crystals when the crystal size is small enough (e.g., below about 15 nm for iron oxide).

[0126] The term "flash point" for a liquid refers to the lowest temperature at which it can be ignited in liquid or vapor form. For example, solvents with a flash point of 2 degrees Celsius are easily ignited in almost any normal workplace and therefore require precautions, while solvents with flash points above 50 degrees Celsius only pose an ignition risk under specific extreme conditions. The table below lists the flash points of some common solvents.

[0127]

[0128] Throughout the instruction manual, these abbreviations have the following meanings:

[0129]

[0130] method

[0131] In Figure 1, the sample is first subjected to protein separation (e.g., immunoprecipitation) to separate any proteins in the sample. Then, any separated proteins can undergo various downstream processes. An aqueous nucleic acid purification buffer containing a polar aprotic solvent and a solid support containing surface silanol groups (e.g., magnetic beads) are then added to the sample. The resulting DNA-bound solid support is then washed and eluted to form free DNA, which is then subjected to various downstream processes. A solid support containing surface carboxyl groups (e.g., magnetic beads) is then added to the sample. The resulting RNA-bound solid support is then removed, washed, and eluted to form free RNA, which is then subjected to various downstream processes.

[0132] Step 0 of Figure 2 (optional, not depicted): Enrich the protein and / or nucleic acid source to be isolated (e.g., circulating tumor cells (CTCs)) using techniques known in the art, or using Dynabeads, for example, conjugated with CTC or exosome-specific antibodies. TM Exosome separation is performed using magnetic beads or SAX / charge conversion beads for general exosome enrichment.

[0133] Step 1 of Figure 2: Protein isolation from a liquid biopsy sample matrix that has been depleted of cells. Alternatively, proteins can be similarly isolated from exosome lysates, such as those from single cells or tumors, or from any other protein and nucleic acid source suitable for immunoprecipitation of protein targets (e.g., plasma, serum, urine, etc.). Proteins are isolated from the sample by adding target-specific antibody-conjugated beads directly to the sample matrix. The bead / protein complex is transferred to a new tube for further processing until the captured proteins are ready for downstream readout (e.g., mass spectrometry, immunoassay, etc.). Non-targeted protein isolation can be performed instead of or attached to targeted protein isolation. Several different proteins can also be isolated by repeating the targeted isolation steps using solid supports with different affinities for the respective targets (e.g., beads conjugated with different target-specific antibodies). Optionally, after these sequential targeted protein isolations or single targeted protein isolations, non-targeted protein isolation can be performed to precipitate the remaining proteins.

[0134] Step 2 in Figure 2: By adding ProK, lysis buffer, and Dynabeads TM MyOne TM DNA separation is performed using silanes and aprotic solvents to trigger DNA binding to beads. The bead / DNA complex is then transferred to a new test tube for downstream processing.

[0135] Step 3 in Figure 2: Adding Dynabeads to the DNA-free lysate TM MyOne TM Carboxylic acids are used for RNA separation to facilitate RNA binding. The sample matrix is ​​then removed, and further processing continues.

[0136] Step 4 of Figure 2: Adding Dynabeads to the DNA-free lysate TM MyOne TM Carboxylic acids are used for RNA separation to facilitate RNA binding. The sample matrix is ​​then removed, and further processing continues.

[0137] Buffer and solvent

[0138] Methods for isolating nucleic acids typically involve exposing the nucleic acids to an aqueous solution, followed by precipitation onto a solid support. Examples of such methods are provided in WO 2012 / 069660. A crucial component of this method is the buffer solution used to precipitate the nucleic acids onto the solid support.

[0139] Compared to purification buffers based on, for example, alcohols and polyols used in the prior art, the nucleic acid purification buffers containing polar aprotic solvents used in the method of the present invention offer numerous advantages. The inventors have demonstrated that purification buffers containing polar aprotic solvents facilitate differential binding of DNA and RNA from the same sample to solid supports (e.g., beads) functionalized with silanols and carboxyl groups, respectively, without the need to change the purification buffer. This significantly simplifies and reduces the complexity and cost of workflows for the sequential separation of total DNA, RNA, and optionally proteins for multi-omics readouts. Furthermore, polar aprotic solvents are generally non-flammable and low in volatility, characteristics not found in common alcohols. This provides benefits such as improved safety in handling and transportation, for example, allowing the buffer to be packaged in ready-to-use form in sealed containers and boxes for transport. Low volatility also reduces the degree of change in component concentration over time. Polar aprotic solvents typically have relatively low viscosity (e.g., less than 50 cP at 20°C and 1 atm), which enables a good level of accuracy and ease of measurement and dispensing of the buffer. Buffer solutions also offer other benefits such as being relatively environmentally friendly (containing biodegradable and renewable components), meeting health and safety standards, and being compatible with downstream nucleic acid processing / analysis procedures such as amplification or mass spectrometry.

[0140] Biorenewable solvents are derived from renewable, sustainable bio-based materials, thus significantly reducing their environmental impact. For example, biorenewable solvents can be >50% bio-based, as demonstrated by carbon testing or by tracking bio-based content at all manufacturing stages.

[0141] Viscosity can be measured according to standard methods, such as those according to the OECD (2012). Test No. 114: Viscosity of Liquids , OECD Guidelines for the Testing of Chemicals, Section1, OECD Publishing, Paris, https: / / doi.org / 10.1787 / 9789264185180-en The method, the content of which is incorporated into this article by reference.

[0142] The polar aprotic solvent may be or may contain N-butylpyrrolidone-2-one (Tamisolve™):

[0143] .

[0144] The polar aprotic solvent may be or may contain dihydro-L-glucanone (Cyrene™):

[0145] .

[0146] The polar aprotic solvent may be or may contain dipropylene glycol dimethyl ether (Proglyde™):

[0147] .

[0148] Exemplary release agents include guanidine salts (such as GuSCN and GuHCl), urea, etc. Surfactants can be selected from nonionic surfactants (such as Triton™ X-100, DDM, digitonin, Tween™ 20, Tween™ 80, Ecosurf™, Brij®, etc.), anionic surfactants (such as sodium lauryl sulfate, deoxycholate, cholate, sodium sarkosyl, etc.), and zwitterionic surfactants (such as CHAPS, Zwittergent® 3-14, etc.). Buffers can be any suitable buffer that provides buffering in a pH range of about 5 to about 9. Examples of suitable buffers include Tris, Trizma, citrate, phosphate, Tricine, TAPS, PBS, acetate, borate, HEPES, Bicine, MOPS, CHES, carbonate, etc., and mixtures thereof; and buffers may also contain other components such as polyethylene glycol (PEG), tetraethylene glycol (TEG), and linear polyacrylamide (LPA). Enzymes are typically proteins or other degrading enzymes, such as proteinase K or lysozyme. Exemplary inorganic salts include metal halides, such as MgCl2, NaCl, LiCl, etc.

[0149] The terms nucleic acid purification buffer and (nucleic acid) binding buffer are used interchangeably.

[0150] Lysis or lysis / binding buffers typically contain a dissociative agent. Dissociative agents denature macromolecules (such as proteins and nucleic acids) in biological materials. They also disrupt membrane lipids. Therefore, the role of the dissociative agent in the lysis solution is to reduce enzyme activity and promote induced cell lysis. Any suitable dissociative agent can be used in a lysis or lysis / binding buffer. Any suitable combination of dissociative agents can be used in a lysis or lysis / binding buffer. For example, in some embodiments, the dissociative agent is selected from guanidine salts (e.g., guanidine isothiocyanate or guanidine hydrochloride), lithium perchlorate, lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and combinations thereof. In some embodiments, the dissociative agent is selected from guanidine salts (e.g., guanidine isothiocyanate, guanidine isocyanate or guanidine hydrochloride), lithium perchlorate, lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, thiocyanate, urea, and combinations thereof. A chaotropic agent (or chaotrope) is present in the lysis or lysis / binding buffer at a concentration sufficient to denature macromolecules in biological material and / or induce cell lysis. In some embodiments, the lysis or lysis / binding buffer contains a guanidine salt at a concentration of at least about 2 M, at least about 3 M, such as at least about 3.5 M or about 4.0 M, for example, about 3 M to 6 M. In some embodiments, the lysis or lysis / binding buffer contains a guanidine salt at a concentration of at least about 1 M, at least about 1.5 M, at least about 2 M, at least about 3 M, such as at least about 3.5 M or about 4.0 M, for example, about 1 M to 6 M. In a preferred embodiment, the lysis buffer contains guanidine (iso)thiocyanate at a final concentration of about 1.0 M to 4.5 M, 1.5 M to 3.5 M, or 3.5 M to 4.5 M, or guanidine hydrochloride at a concentration of about 3.5 M to 4.5 M, for example, about 4.0 M. The final concentrations during lysis and binding steps differed due to the addition of the sample and trigger, respectively.

[0151] Lysis or lysis / binding buffers typically contain surfactants, such as nonionic surfactants or detergents, among which poly(ethylene oxide)-containing surfactants may be mentioned. The role of detergents in lysis solutions is to disrupt cell and organelle membranes (e.g., lyse cells and organelles) and denature proteins in biological materials. Therefore, detergents are used to facilitate the release of nucleic acids from cells and other entities (e.g., viruses) in biological materials. Any suitable detergent (e.g., a nonionic detergent) can be used in lysis or lysis / binding solutions. For example, in some embodiments, detergents are selected from sodium lauroyl sarcosinate (sodium dodecyl sarcosinate), sodium dodecyl sulfate (SOS), polyoxyethylene-20-sorbitan monolaurate (Tween®-20)™, Ecosurf™, CHAPS, Brij®, Zwittergent® 3-14, and combinations thereof. In preferred embodiments, detergents are selected from polyoxyethylene-20-sorbitan monolaurate (Tween®-20). The detergent concentration in the lysis solution is sufficient to disrupt cell membranes and organelle membranes (e.g., lyse cells and organelles), disrupt viral envelopes and / or capsids, and / or denature proteins in biological material. In some embodiments, the detergent is present at concentrations such as about 0.5% w / v to 5.0% w / v, for example about 0.75% w / v to 4.5% w / v, about 1.0% w / v to 4.0% w / v, about 1.5% w / v to 3.0% w / v, such as about 1.75% w / v to 2.25% w / v, for example about 2.0%. In some preferred embodiments, the detergent is sodium lauroyl sarcosinate (sodium dodecyl sarcosinate) or polyoxyethylene-20-sorbitan monolaurate (Tween®-20) at concentrations as defined above.

[0152] The lysis / binding buffer can be any buffer known for this purpose, such as a buffer containing a high concentration of a dissociating agent (e.g., guanidine salt or urea) and a surfactant (e.g., polyoxyethylene-20-sorbitan monolaurate (Tween®-20)). For example, a preferred lysis buffer is a buffer containing a high concentration of a detergent, guanidine isothiocyanate (GTC).

[0153] In some cases, the lysis or lysis / binding buffer may contain citrate buffer, such as sodium citrate or potassium citrate, or may contain one or more chelating agents. Such chelating agents function to isolate divalent cations, which is essential for enzymes that act on nucleic acids, such as DNases and RNases. Therefore, the role of the chelating agent is to inhibit or prevent the degradation of nucleic acids in the sample. Thus, chelating agents include divalent cationic chelating agents, such as EDTA (ethylenediaminetetraacetic acid). The chelating agent can be present in the lysis solution at a concentration sufficient to inhibit nucleic acid-degrading enzymes (i.e., in samples containing biological material), such as about 5 mM to 50 mM, such as about 10 mM to 40 mM, or about 15 mM to 30 mM, such as about 20 mM. Therefore, in some embodiments, the lysis solution contains EDTA at a concentration as defined above. Instead of or in addition to one or more chelating agents, one or more other DNase and / or RNase inhibitors known in the art may be present in the lysis / binding buffer.

[0154] Lysis or lysis / binding buffers may also contain one or more reducing agents. When present in a lysis buffer, the reducing agent acts to reduce disulfide bonds in proteins of biological material. Suitable reducing agents are well known in the art and are selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), β-mercaptoethanol (β-ME), and combinations thereof. The use of TCEP is particularly advantageous because it has high stability and activity at room temperature, thus facilitating the production of lysis buffers with improved activity and long-term storage (e.g., for large-scale production of commercial products). Therefore, in some preferred embodiments, the reducing agent is TCEP.

[0155] When included in a lysis or lysis / binding buffer, the reducing agent can be present at a concentration sufficient to reduce disulfide bonds in proteins of biological material. In some embodiments, the reducing agent can be present at a concentration of about 1 mM to 20 mM, such as about 2 mM to 19 mM, 3 mM to 18 mM, 4 mM to 17 mM, or about 5 mM to 16 mM, such as about 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM, preferably about 10 mM. However, in some embodiments, higher amounts of the reducing agent can be used, such as about 20 mM to 150 mM, such as about 25 mM to 125 mM, or about 30 mM to 100 mM, such as about 80 mM.

[0156] Furthermore, the lysis or lysis / binding buffer suitable for the methods and uses of this invention may contain a “nucleic acid carrier” for increasing the concentration of nucleic acids in the sample. In some embodiments, this may promote nucleic acid aggregation induced by a precipitant and adsorption onto a solid support (e.g., silica-coated magnetic beads). The nucleic acid carrier is typically a polymer, such as nucleic acid or polysaccharide. For example, the 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 combinations thereof. In some instances, 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). Therefore, in some preferred embodiments, the nucleic acid carrier may be glycogen.

[0157] When a nucleic acid carrier is included in the lysis or lysis / binding buffer, the nucleic acid carrier can be present at a concentration sufficient to increase the efficiency of nucleic acid recovery from biological materials (i.e., sufficient to increase the adsorption of nucleic acids on solid supports). In some embodiments, the nucleic acid carrier can be present in the lysis buffer at a concentration of about 0.1 mg / ml to 5 mg / ml, such as about 0.2 mg / ml to 4.0 mg / ml, 0.3 mg / ml to 4.0 mg / ml, 0.4 mg / ml to 3.0 mg / ml, or about 0.5 mg / ml to 3.0 mg / ml, for example about 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, or 1.5 mg / ml, preferably about 1.0 mg / ml.

[0158] The lysis step may also utilize other components, such as proteases, for example, proteinase K. In some cases, proteases can improve nucleic acid extraction efficiency. Therefore, lysis can be performed by combining cells, viruses, or another biological structure with a dissociation agent (e.g., guanidine salt or urea), a surfactant (e.g., a nonionic surfactant, such as a polyoxyethylene surfactant), and a protease (e.g., proteinase, such as proteinase K). In some embodiments, the lysis buffer may contain a protease (e.g., proteinase). Alternatively, in some embodiments, the protease (e.g., proteinase) may be added to the sample after it has been contacted with the lysis buffer but before it has been contacted with the binding buffer or purification buffer. In some embodiments, the protease (e.g., proteinase) may be added while the sample is in contact with a solid support (e.g., a suspension of magnetic particles).

[0159] In the nucleic acid binding step of a standard nucleic acid isolation protocol, the lysis / binding buffer is typically supplemented with isopropanol, such as 50% isopropanol, or may be supplemented with ethanol, to facilitate nucleic acid capture on a solid support. However, this is not ideal, for example, because isopropanol is volatile and flammable.

[0160] Real-time detection

[0161] Point-of-care testing (POC) can be performed using point-of-care instruments. POC can include the use of cartridges or microfluidic chips. A cartridge may include compartments containing one or more of the following: a solid support, an aqueous medium containing a polar aprotic solvent (e.g., an aqueous nucleic acid purification buffer as disclosed herein), a wash buffer, and an elution buffer. The cartridge may also contain the necessary microfluidics for transferring reagents between compartments to perform the nucleic acid processing methods described herein. Such cartridges are typically transported and stored in ready-to-use form, and therefore are advantageous if the reagents contained therein (such as aqueous media containing polar aprotic solvents, and / or buffers) are non-flammable.

[0162] Exemplary point-of-care detection methods may include introducing a sample into a cartridge or microfluidic chip, and performing the following steps on the cartridge:

[0163] Samples containing nucleic acids are exposed to an aqueous medium containing a polar aprotic solvent in the presence of a solid support.

[0164] Nucleic acid is precipitated onto a solid support, thereby providing a solid support for nucleic acid binding;

[0165] The nucleic acid-bound solid support was washed with washing buffer; and

[0166] The solid support for binding nucleic acids is contacted with elution buffer, thereby separating the nucleic acids from the solid support to provide isolated nucleic acids;

[0167] The nucleic acid is then subjected to one or more additional processes. Examples of additional processes that may be associated with point-of-care testing include, but are not limited to, detection, quantification, cloning, analysis, epigenetic analysis, sequencing, amplification, research, transfection, hybridization, cDNA synthesis, size separation, chromatography, and mass spectrometry. The sample may be a supernatant from one or more targeted and / or non-targeted protein separations.

[0168] Examples of instruments suitable for use in the methods according to this disclosure are provided in US 9,752,182 B2 and US 2016 / 0016171 A1, the contents of which are incorporated herein by reference.

[0169] Automated nucleic acid analysis platform

[0170] The advantages of automated nucleic acid analysis platforms include the ability to analyze large numbers of samples in parallel within a shortened timeframe, eliminating the risk of manual processing errors during analysis, and requiring minimal operator intervention time. The kits of this invention can be used with automated liquid handling and pipetting systems, as well as with automated, multi-purpose, high-throughput integrated laboratory systems that include downstream processing capabilities. Exemplary automated protein and / or nucleic acid analysis platforms used in this invention may include, but are not limited to, liquid handling and automated systems such as the KingFisher™ system, DreamPrep™ NAP workstation (TECAN), Fluent™ Automated Workstation (TECAN), Microlab Prep, NIMBUS, STAR, or VANTAGE pipetting platform (Hamilton Company). Furthermore, compatible automated systems can provide downstream processing (such as genotyping or diagnostic assays), such as the COR™ MX / PX or GX system (Beckton Dickinson) and the COBAS™ 5800 system (Roche Diagnostics). Automated nucleic acid analysis platforms can be point-of-care instruments.

[0171] Point-of-care testing instruments. Point-of-care testing instruments can be used for the detection of clinical biomarkers and / or drugs and / or pathogens and / or biological warfare agents and / or genetic diseases. Pathogen detection or biological warfare agent detection may include the detection of viral and / or bacterial and / or fungal proteins and / or nucleic acids. Exemplary point-of-care testing instruments include ePlex. ®The system (available from GenMark Diagnostics, Inc., Carlsbad, California, USA) and Solana ® Instruments are available from Quidel in San Diego, California, USA. Currently available instruments also include the Roche cobas® 8000 modular analyzer series, the VIDAS® system from bioMerieux, or the ARCHITECT analyzer from Abbott. Siemens, DiaSorin, and Ortho Clinical Diagnostics also offer multi-target analyzer systems. KRYPTOR TM The analyzer is sold by Thermo Fisher. Further examples of point-of-care instruments are provided in US 9,752,182 B2 and US 2016 / 0016171 A1, the contents of which are incorporated herein by reference.

[0172] Additional Examples

[0173] This invention and disclosure also include the subject matter of the following provisions:

[0174] 1. A method for sequentially separating DNA and RNA from a sample, the method comprising steps a) to d):

[0175] a) In the presence of an aqueous nucleic acid purification buffer, the sample is contacted with a solid support containing surface silanol groups to provide a solid support for DNA binding;

[0176] b) Remove the solid support containing the DNA from the sample;

[0177] c) In the presence of an aqueous nucleic acid purification buffer, contact the sample with a solid support containing surface carboxyl groups to provide a solid support for RNA binding; and

[0178] d) Remove the solid support containing the RNA from the fluid sample.

[0179] The aqueous nucleic acid purification buffer contains a polar aprotic solvent.

[0180] 2. The method according to Clause 1, wherein the polar aprotic solvent is selected from one or more of the following: dihydro-L-glucosinolate, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, and N-formylmorpholine.

[0181] 3. The method according to Clause 1 or Clause 2, wherein the polar aprotic solvent is N-butylpyrrolidone-2-one.

[0182] 4. The method according to any of the preceding clauses further includes adding the aqueous nucleic acid purification buffer to the sample prior to steps a) to d).

[0183] 5. The method according to any of the preceding clauses, wherein the method is a method for sequentially separating proteins, DNA and RNA from the sample, the method further comprising separating proteins prior to step a).

[0184] 6. The method according to Clause 5, wherein protein separation comprises non-targeted protein separation and / or affinity protein separation, optionally wherein non-targeted affinity protein separation comprises contacting the sample with a solid phase having weak to strong anionic or cationic surface chemistry (which enables ion exchange), and further optionally wherein the affinity protein separation is immunoprecipitation.

[0185] 7. The method according to Clause 6, wherein the affinity protein separation comprises:

[0186] The sample is contacted with a protein affinity reagent immobilized on a solid support to provide a protein-binding solid support; and

[0187] Remove the protein-bound solid support from the fluid sample.

[0188] 8. The method according to any of the preceding clauses, wherein the method further includes a pyrolysis step.

[0189] 9. The method according to Clause 8, wherein the lysis step is performed before or during step a); optionally, the lysis step is performed before affinity protein separation.

[0190] 10. The method according to clause 8 or 9, wherein the lysis step includes adding lysis buffer to the sample.

[0191] 11. The method according to any of the preceding clauses further comprises contacting the sample with proteinase K prior to step a), provided that the proteinase K is not added until after the (optional) affinity protein has been isolated.

[0192] 12. The method according to any of the preceding clauses, wherein the solid support comprises (optionally monodisperse) beads, and further optionally wherein the (optionally monodisperse) beads are magnetic.

[0193] 13. The method according to any of the preceding clauses, wherein step b) further comprises b2) washing the DNA-bound solid support with a washing buffer, optionally wherein the washing buffer is or contains the purification buffer.

[0194] 14. The method according to any of the preceding clauses, wherein step b) further comprises b3) contacting the solid support bound to the DNA with an elution buffer to separate the DNA from its solid support, wherein optionally the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

[0195] 15. The method according to Clause 14, wherein the isolated DNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from microarrays, qPCR, dPCR, and next-generation sequencing.

[0196] 16. The method according to any of the preceding clauses, wherein step d) further includes d2) washing the RNA-bound solid support with a washing buffer, optionally wherein the washing buffer is or contains the purification buffer.

[0197] 17. The method according to any of the preceding clauses, wherein step d) further includes d3) contacting the solid support to which the RNA is bound with an elution buffer to separate the RNA from its solid support, wherein optionally the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

[0198] 18. The method according to Clause 17, wherein the isolated RNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from microarrays, qPCR, dPCR, and next-generation sequencing.

[0199] 19. The method according to any of the preceding clauses, wherein the polar aprotic solvent is present in an amount of at least about 2% by weight, optionally wherein the polar aprotic solvent is present in an amount of no more than about 80% by weight.

[0200] 20. The method according to any of the preceding clauses, wherein the purification buffer further comprises one or more of the following: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, an antifoaming agent, or a combination thereof.

[0201] 21. The method according to any of the preceding clauses, wherein the purification buffer is a binding buffer and / or a washing buffer, optionally wherein the washing buffer comprises a 1.1 to 5-fold dilution of the binding buffer.

[0202] 22. The method according to any of the preceding clauses, wherein the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, acetone or dimethyl sulfoxide.

[0203] 23. The method according to any of the preceding clauses, wherein the DNA is cfDNA and / or ctDNA and / or the RNA is cfRNA and / or ctRNA.

[0204] 24. The method according to any of the preceding clauses, wherein the sample is a biological or environmental sample.

[0205] 25. The method according to any of the preceding clauses, wherein the sample is a biological sample, optionally wherein:

[0206] The biological sample is a fluid biological sample (e.g., a liquid biopsy).

[0207] The biological sample is a cell-based sample;

[0208] The biological sample is a single-cell-based sample, CTC, exosome, tissue biopsy material, or FFPE;

[0209] The biological sample includes CTC, the DNA includes gDNA, and the RNA includes total RNA; or

[0210] The biological sample includes exosomes, the DNA includes cfDNA, and the RNA includes one or more of the following: miRNA, mRNA, and snRNA.

[0211] 26. A reagent kit comprising:

[0212] A first solid support, the first solid support comprising surface silanol groups;

[0213] A second solid support, the second solid support comprising surface carboxyl groups; and

[0214] An aqueous nucleic acid purification buffer, wherein the aqueous nucleic acid purification buffer contains a polar aprotic solvent.

[0215] 27. The kit according to Clause 26, wherein the polar aprotic solvent is selected from one or more of the following: dihydro-L-glucanone, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, and N-formylmorpholine, optionally wherein the polar aprotic solvent is N-butylpyrrolidone-2-one.

[0216] 28. The kit according to Clause 26 or 27 further comprises a third solid support containing an immobilized protein affinity reagent.

[0217] 29. The kit according to any one of clauses 26 to 28 further comprises a lysis buffer.

[0218] 30. The kit according to any one of clauses 26 to 29, further comprising proteinase K.

[0219] 31. The kit according to any one of Clauses 26 to 30, wherein the first solid support and / or the second solid support and / or (optionally) the third solid support comprises (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.

[0220] 32. The kit according to any one of clauses 26 to 31, further comprising at least one washing buffer, optionally wherein the at least one washing buffer comprises the purification buffer.

[0221] 33. The kit according to any one of clauses 26 to 32 further comprises an elution buffer, wherein optionally the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

[0222] 34. The kit according to any one of Clauses 26 to 33, wherein the nucleic acid purification buffer further comprises the features defined in any one of Clauses 19 to 22.

[0223] 35. Use of the kit according to any one of Clauses 26 to 34 in an automated nucleic acid analysis platform.

[0224] 36. A nucleic acid analysis device, comprising:

[0225] Automated nucleic acid analysis platform; and

[0226] The kit according to any one of clauses 26 to 34,

[0227] The automated nucleic acid analysis platform includes a portion configured to accommodate the solid support.

[0228] 37. The nucleic acid analysis device according to Clause 36, wherein the automated nucleic acid analysis platform is a point-of-care testing instrument, optionally wherein the point-of-care testing instrument is suitable for cancer detection.

[0229] Example

[0230] Example 1: Protocol for sequentially extracting circulating tumor cells or exosomes, along with proteins, ctDNA, and ctRNA, from equal volumes of liquid biopsy samples.

[0231] The efficiency of exemplary purification buffers containing polar aprotic solvents for nucleic acid purification was evaluated. To investigate the efficiency of these buffers in triggering the binding of nucleic acids to silanol or carboxylic acid-functionalized surfaces, a 120 bp dsDNA fragment (1 e6 copy) and MS2 RNA (1.5 e6 copy) were spiked into 200 µL of human plasma prior to sample processing, and then processed according to the protocol described below. The eluted samples were then analyzed by qPCR to quantify the 120 bp DNA recovery, or by RT-qPCR to quantify the MS2 RNA recovery. Positive controls were treated with the same aqueous NA purification buffer (containing isopropanol, not a polar aprotic solvent). Purification was performed on a KingFisher instrument.

[0232] Reagents:

[0233] Dynabeads™ MyOne Silane Magnetic Beads (Thermo Fisher Scientific).

[0234] Carboxylic acid magnetic beads: Dynabeads™ MyOne™ Carboxylic Acid (Thermo Fisher Scientific).

[0235] Aqueous NA purification buffer containing guanidine salts and Tween.

[0236] Trigger: A nonprotic solvent used for nucleic acid purification buffers, as shown in Table 1 of the examples. Isopropanol (IPA) was used as an alternative trigger for the positive control.

[0237] Wash Buffer 1: 50% Silane Rare Target (NA) purification buffer and 50% IPA. Note that if the 50% IPA used in the trigger is replaced with approximately 50% aprotic solvent, similar results are expected.

[0238] Wash buffer 2: 70% aqueous ethanol solution. Note that if the 70% ethanol is replaced with approximately 70% of the triggering agent, similar results are expected.

[0239] Elution buffer: 10 mM Tris pH 8.

[0240] LB buffer contains guanidine salts, ionic or nonionic detergents (e.g., Tween, such as Tween20), pH 5-9.

[0241] Proteinase K, 20 mg / mL.

[0242] Table 1: Trigger parameters used for trigger testing

[0243]

[0244] Consumables and equipment:

[0245] Magnetic Separator: DynaMag™-2 Magnet, SKU 12321D (Thermo Fisher Scientific). This device provides a support for microtubes with a built-in magnet at the bottom, effectively separating magnetic beads from the supernatant.

[0246] 1.5 mL microtube.

[0247] Tube rollers / rotators.

[0248] Microtube heating block (preheated to the required pyrolysis and / or elution temperature).

[0249] Microtube benchtop centrifuge.

[0250] 1000 µL, 200 µL and 100 µL pipettes.

[0251] Timer.

[0252] Hot mixer / shaker.

[0253] Optional equipment for efficient manual processing:

[0254] Laboratory vacuum pumps used to remove supernatant;

[0255] Disposable glass Pasteur pipette with a long capillary tip.

[0256] program:

[0257] 1. Protein isolation (optional): Steps a) through g) provide an exemplary workflow for immunoprecipitation of liquid biopsies or soluble proteins:

[0258] a) Dilute the sample at 0.5 vol in PBS or PBST (pH 7.4) containing 5 mM EDTA or HEPES (pH 7.4) buffer containing detergent (such as Tween-20, as provided in the Dynabeads™ Co-immunoprecipitation Kit (Thermo Fisher Scientific catalog number 14321D)).

[0259] b) Add the sample containing the antigen (Ag) (usually 100-1,000 µl) to the tube containing the Dynabeads conjugate antibody and gently aspirate to resuspend the Dynabeads.

[0260] c) Incubate at room temperature by rotation for 10 minutes to allow Ag to bind to the Dynabeads-Ab complex. Note: Depending on the antibody affinity, the incubation time may need to be increased to achieve optimal binding.

[0261] d) Place the tube on the magnet. Transfer the supernatant to a clean tube for further extraction of DNA / RNA or both (starting with optional proteinase K digestion in step 3).

[0262] e) For each wash, wash the Dynabeads-Ab-Ag complex three times with 200 µL to 500 µL of wash buffer (e.g., PBST, or NH4OAc-based buffer, with or without detergent, as provided in the Dynabeads™ Co-immunoprecipitation Kit (Thermo Fisher Scientific catalog number 14321D), depending on the downstream assay). Between each wash, separate on a magnet, remove the supernatant, and resuspend by gentle pipetting.

[0263] f) Resuspend the Dynabeads-Ab-Ag complex in 100 µL to 500 µL of wash buffer and transfer the bead suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall. (At this point, the Ag bound to the beads can also be used for immunoassay readout).

[0264] g) Optionally, the bound protein is eluted in a desired volume of 0.5 M NH4OH plus 0.5 mM EDTA (for mass spectrometry analysis) or in a desired volume of 200 mM glycine (pH 2.8) for standard gel analysis.

[0265] 2. Allow the buffer solution to reach room temperature before use. Add the silane... Magnetic beads Vortex for 20 seconds to resuspend the beads. Before using in step 6, rotate the bottle on a roller / rotator for 20 minutes at room temperature.

[0266] 3. Add 200 µL of serum / plasma / UTM (universal transfer medium) sample or the supernatant from step 1 d) to a 1.5 mL tube.

[0267] 4. Add 50 µL Proteinase K Accompanied by brief pulse vortexes.

[0268] 5. Add 300 µL LB buffer Then, perform a brief pulsed vortex and then incubate at room temperature for 10 minutes. Incubation for 10 minutes at 900 rpm in a hot mixer / shaker will be sufficient.

[0269] 6. Add 25 µL Dynabeads TM MyOne TM Silane (1 mg) Then, a brief pulse vortex occurs.

[0270] 7. Add 150 µL Trigger Gently add to the lysate and mix under brief pulsed vortexing, then incubate the tube on a vortex mixer / roller for 10 minutes at room temperature. Incubation for 10 minutes at 1050 rpm in a hot mixer / shaker will be sufficient.

[0271] 8. Place the tube on the DynaMag™-2 magnet. Allow the beads to adhere to the magnet for 1 to 2 minutes, or until the beads are completely removed.

[0272] 9. Carefully remove using a 1000 µL pipette or vacuum pump. pyrolysis products Then the pyrolysis product is subjected to step 25.

[0273] 10. Washing buffer 1. First wash: After removing the tube from the magnet, add 800µl Washing buffer 1 Vortex until the bead is completely resuspended. Place the tube on a DynaMag™-2 magnet and allow the bead to adhere to the magnet for 1 to 2 minutes.

[0274] 11. Carefully remove using a 1000 µL pipette or vacuum pump. Washing buffer 1 And discard it.

[0275] 12. Washing buffer 1. Second wash: By repeating steps 9-10, as described in step 9, using the same volume of... Washing buffer 1 Perform a second wash.

[0276] 13. Washing buffer 2. First wash: After removing the tube from the magnet, add 500 µL Washing buffer 2 Mix by vortexing / pipetting until the beads are completely resuspended. Transfer the bead solution to a new 1.5 mL microtube placed on a DynaMag™-2 magnet using a 1000 µL pipette.

[0277] 14. Allow the beads to adhere to the magnet for 1 minute. Carefully remove them from the 1.5 mL tube using a pipette with a 1000 µL tip or a vacuum pump. Washing buffer 2 And discard it.

[0278] 15. Washing buffer 2. Second wash: Repeat steps 13 and 14 (but do not transfer the bead solution to a new tube).

[0279] 16. To remove trapped fluid between the beads, tap the holder / magnet with the tube on the worktable surface with moderate force 3 to 5 times. Use a small pipette to aspirate the last drop and discard it. Remove all fluid before elution.

[0280] 17. Add 100 µL Elution buffer .

[0281] 18. Vortex for 20 seconds to 1 minute until the bead precipitate is completely resuspended, then briefly centrifuge (1-second pulse), and collect the bead suspension. Take care not to allow the beads to settle.

[0282] 19. Incubate the bead suspension in a preheated block at 80°C for 5 minutes. Mixing at 1500 rpm for 5 minutes at 80°C in a hot mixer / shaker will be sufficient.

[0283] 20. After vortexing for 15 seconds, briefly centrifuge the bead suspension (1-second pulse).

[0284] 21. Place the tube on the DynaMag™-2 magnet. Allow the beads to adhere to the magnet for 15 seconds.

[0285] 22. Transfer the eluted DNA to a new 1.5 mL tube and store at an appropriate temperature (e.g., 4°C to -80°C).

[0286] 23. Take 100 µL DynabeadsTM MyOne TM Carboxylic acid (1 mg) Add to the pyrolyte from step 9 and mix under a brief pulsed vortex.

[0287] 24. Incubate the tubes on a rotator / roller for 10 minutes at room temperature. When using a hot mixer / shaker, 10 minutes at 900 rpm will be sufficient.

[0288] 25. For those containing Dynabeads MyOne Carboxylic Acid 100 µL of the lysate (1 mg) was processed in steps 8 to 21.

[0289] 28. Transfer the eluted RNA to a new 1.5 mL tube and store at an appropriate temperature (e.g., 4°C to -80°C).

[0290] Figure 3 shows the results for TamiSolve™. When used with silane rare target NA purification buffer, TamiSolve™ produced nearly 100% DNA target recovery, while less than 4% of RNA targets bound to the silane beads. Subsequent binding of RNA to Dynabeads MyOne carboxylic acid yielded approximately 77% RNA target recovery.

[0291] Example 2: Changes in washing buffer

[0292] Repeat the protocol for sequential DNA and RNA extraction as described in Example 1 (positive control and Examples 2a through 2f). Table 2 below provides the trigger, wash buffer 1, and wash buffer 2 used in each replicate, as well as the DNA and RNA purification rates after the first and second elutions. While this does not affect the initial binding, the final recovery rate may be affected by the choice of wash buffer 1 and wash buffer 2.

[0293] Table 2: Changes in Washing Buffer

[0294]

[0295] 50% Silane Rare Target NA Purification Buffer

[0296] Volume, water

[0297] The data in Table 2 confirms that TamiSolve™ will trigger a large amount of DNA and Dynabeads TM MyOne TM silicon alkyl The data also showed the binding of RNA to Dynabeads™ MyOne™ carboxylic acid and the binding of a large amount of RNA to Dynabeads™ MyOne™ carboxylic acid. Furthermore, the data indicated that good DNA and RNA purification rates could be achieved even with changes to wash buffer 1 and wash buffer 2.

[0298] Example 3: Ensuring consistency across different silane beads

[0299] Repeat the protocol for sequential DNA and RNA extraction as described in Example 1 (positive control and Examples 3a through 3d). Two different batches were tested in parallel. Dynabeads TM MyOne TM silane (Bead batch a and bead batch b). Table 3 below shows the triggering agent, wash buffer 1, and wash buffer 2 used in each replicate, as well as the DNA and RNA purification rates after the first and second elutions. Similar results were obtained for both bead batches.

[0300] Table 3: Comparison of results between two batches of silane beads

[0301]

[0302] 50% Silane Rare Target NA Purification Buffer

[0303] Volume, water

[0304] Example 4: Sequential purification of proteins, DNA, and RNA

[0305] The plasma sample was spiked with 500 pg / mL IL-6 protein and 5,000,000 x 10 6 120 bp DNA copies / mL. For each extraction, use 200 µl of plasma containing IL-6 and 120 bp DNA, and pipette into a KingFisher plate. Add 20 µg of Dynabeads™ MyOne™ Tosylactivated conjugated with anti-IL-6 antibody, mix slowly for 20 minutes, and then place on a magnet. Aspirate the supernatant and use it to isolate nucleic acids.

[0306] The beads were transferred to 200 µL of assay buffer (TBST-BSA) and resuspended. 100 µL was transferred to a white, flat-bottomed 96-well plate. 50 µL (0.1 µg / mL) of acrid ester (AE)-labeled detection antibody was added, and the plate was incubated on a shaker at 1000 rpm at 37°C. The beads were washed three times with 300 µL of TBS using a plate washer with a magnet. Finally, the beads were resuspended in 50 µL of TBST, and the signal was read using Flash on a Varioskan LUX. A standard curve for IL-6 was generated in parallel to quantify the amount of precipitated protein.

[0307] After adding MS2 RNA (1.5 x 10⁻⁶) 6 After copying, the supernatant is subjected to DNA and RNA separation (as described in Example 1).

[0308] The control workflow used IPA as the trigger for precipitation onto the beads, while the experimental workflows (Examples 4a and 4b) used Tamisolve as the trigger, but with different wash buffers. Table 4 below provides the reagents used in each workflow, along with the corresponding protein, DNA, and RNA purification rates after each elution.

[0309] Table 4: Protein purification rate, DNA purification rate, and RNA purification rate

[0310]

[0311] 50% Silane Rare Target NA Purification Buffer

[0312] Volume, water

[0313] The data in Table 4 indicate that polar aprotic solvents (e.g., TamiSolve™) can trigger a wide range of target proteins to react with appropriate solid supports (e.g., with...). Dynabeads™ MyOne™ Tosylactivated The binding of the conjugated IL-6 antibody triggers DNA and RNA binding during subsequent purification without altering the buffer; simply adding appropriate solid support (e.g., for DNA separation) is sufficient. Dynabeads TM MyOne TM silane and for RNA isolation Dynabeads™ MyOne™ Carboxylic Acid ).

Claims

1. A method for sequentially separating DNA and RNA from a sample, the method comprising steps a) to d): a) In the presence of an aqueous nucleic acid purification buffer, the sample is contacted with a solid support containing surface silanol groups to provide a solid support for DNA binding; b) Remove the solid support containing the DNA from the sample; c) In the presence of the aqueous nucleic acid purification buffer, contact the sample with a solid support containing surface carboxyl groups to provide a solid support for RNA binding; and d) Remove the solid support containing the RNA from the fluid sample. The aqueous nucleic acid purification buffer contains a polar aprotic solvent.

2. The method according to claim 1, wherein the polar aprotic solvent has a boiling point of more than 100°C at 1 atm, optionally having a boiling point of more than 150°C at 1 atm.

3. The method according to 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 according to any of the preceding claims, 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 according to any of the preceding claims, 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 according to any of the preceding claims, wherein the polar aprotic solvent has a flash point of at least 50°C.

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

8. The method according to claim 1, wherein the polar aprotic solvent is selected from one or more of the following: dihydro-L-glucosinolate, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, and N-formylmorpholine.

9. The method according to claim 8, wherein the polar aprotic solvent is N-butylpyrrolidone-2-one.

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

11. The method according to any of the preceding claims, wherein the method is a method for sequentially separating proteins, DNA and RNA from the sample, the method further comprising separating the proteins prior to step a).

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

13. The method of claim 12, wherein the affinity protein separation comprises: The fluid sample is brought into contact with a protein affinity reagent immobilized on a solid support to provide a protein-binding solid support; as well as Remove the protein-bound solid support from the fluid sample.

14. The method according to any of the preceding claims, wherein the method further comprises a pyrolysis step.

15. The method of claim 14, wherein the lysis step is performed before 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 according to any of the preceding claims, further comprising contacting the sample with proteinase K prior to step a), wherein the proteinase K is added only after the (optional) affinity protein has been isolated.

18. The method according to any preceding claim, wherein the solid support comprises one or more of the following: particles, resin, box, beads, filter, column, array, membrane, chip, disk or glass slide.

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

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

21. The method according to any of the preceding claims, wherein step b) further comprises b3) contacting the solid support bound to the DNA with an elution buffer to separate the DNA from its solid support, wherein optionally the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

22. The method of claim 21, wherein the isolated DNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from detection, quantification, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical formulation 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 according to claim 23, wherein the isothermal amplification includes loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple substitution amplification (MDA), recombinase polymerase amplification (RPA), strand substitution amplification (SDA), multiple cross substitution amplification (MCDA), signal-mediated RNA amplification (SMART), recombinase-polymerase amplification (RPA), or nucleic acid sequence-based amplification (NASBA).

25. The method of claim 22, wherein the isolated DNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from microarrays, qPCR, dPCR, and next-generation sequencing.

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

27. The method according to any of the preceding claims, wherein step d) further comprises d3) contacting the solid support to which the RNA is bound with an elution buffer to separate the RNA from its solid support, wherein optionally the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

28. The method of claim 27, wherein the isolated RNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from detection, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical formulation 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 includes loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple substitution amplification (MDA), recombinase polymerase amplification (RPA), strand substitution amplification (SDA), multiple cross substitution amplification (MCDA), signal-mediated RNA amplification (SMART), recombinase-polymerase amplification (RPA), or nucleic acid sequence-based amplification (NASBA).

31. The method of claim 27, wherein the isolated RNA is further subjected to one or more additional processes, said one or more additional processes optionally selected from microarrays, qPCR, dPCR, and next-generation sequencing.

32. The method according to any of the preceding claims, wherein the polar aprotic solvent is present in an amount of at least about 2% by weight.

33. The method according to any of the preceding claims, wherein the polar aprotic solvent is present in an amount not exceeding about 80% by weight.

34. The method according to any of the preceding claims, wherein the polar aprotic solvent is present in an amount between about 4% by weight and about 75% by weight.

35. The method according to any preceding claim, wherein the purification buffer further comprises one or more of the following: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, an antifoaming agent, or a combination thereof.

36. The method according to any of the preceding claims, wherein the purification buffer is a binding buffer and / or a washing buffer.

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

38. The method according to any of the preceding claims, wherein the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, acetone or dimethyl sulfoxide.

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

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

41. The method according to any of the preceding claims, wherein the DNA is ctDNA, and / or wherein the RNA is ctRNA.

42. The method according to any of the preceding claims, wherein the sample comprises or is a pretreated or untreated biological sample, environmental sample, or enzymatic reaction mixture.

43. The method according to any preceding claim, wherein the sample is an environmental sample, optionally wherein the environmental sample comprises: Water samples, wastewater samples (optional); soil samples; sediment samples; surface swabs; Airborne samples (such as air filter residue); cosmetics; Food ingredients or food samples; or combinations thereof.

44. The method according to any of the preceding claims, wherein the sample is an enzymatic reaction mixture, optionally wherein the enzymatic reaction mixture comprises an in vitro transcription reaction mixture, a reverse transcription reaction mixture, a second-strand synthesis reaction mixture, an amplification reaction mixture, a library preparation reaction mixture, a restriction enzyme digestion reaction mixture, a nucleic acid assembly reaction mixture, or a barcoding reaction mixture.

45. The method according to any preceding claim, wherein the sample is a biological sample, optionally comprising one or more of the following: blood, bloodstains, umbilical cord blood, blood components (e.g., concentrated platelets), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, erythrocyte sedimentation rate (ESR) leukocytes, serum, plasma, saliva, saliva stains, buccal cells, buccal swabs, semen, semen stains, urine, feces, fecal stains, cigarette butts, chewing gum, formalin. Fixed paraffin-embedded (FFPE) samples, biopsy samples, bone marrow, tissue samples, plant samples, cell lysates, bacterial or yeast cultures, sputum, tears, pharyngeal swabs, oral irrigant, nasopharyngeal swabs, nasopharyngeal aspirates, exhaled fluid, nasal swabs, nasal irrigant, mucus, bronchial aspirates, bronchoalveolar lavage fluid, pleural fluid, tracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, cervical swabs, vitreous fluid, amniotic fluid, and breast milk, optionally in physiological buffer or transfer culture medium.

46. ​​The method according to any preceding 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 a single-cell-based sample, CTC, exosome, tissue biopsy material, or FFPE; The biological sample includes CTC, the DNA includes gDNA, and the RNA includes total RNA; or The biological sample includes exosomes, the DNA includes cfDNA, and the RNA includes one or more of the following: miRNA, mRNA, and snRNA.

47. A reagent kit comprising: A first solid support, the first solid support comprising surface silanol groups; A second solid support, the second solid support comprising surface carboxyl groups; and An aqueous nucleic acid purification buffer, wherein the aqueous nucleic acid purification buffer contains a polar aprotic solvent.

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

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

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

51. The kit according to any one of claims 47 to 50, further comprising proteinase K.

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

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

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

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

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

57. A nucleic acid analysis device, comprising: Automated nucleic acid analysis platform; as well as The kit according to any one of claims 47 to 55, The automated nucleic acid analysis platform includes a portion configured to accommodate the solid support.

58. The nucleic acid analysis device according to claim 57, wherein the automated nucleic acid analysis platform is a point-of-care testing instrument.

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

60. The nucleic acid analysis device according to claim 58, wherein the point-of-care assay instrument is suitable for cancer detection.

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

62. The method according to any one of claims 11 to 46, wherein the protein isolation comprises: In the aqueous nucleic acid purification buffer used for the sequential separation of DNA and RNA, the sample is contacted with an immobilized protein affinity reagent.

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