Nucleic acid preparation

Abstract

Disclosed herein is an aqueous nucleic acid purification buffer comprising a polar aprotic solvent. Also provided herein are using such purification buffers to precipitate nucleic acids from a solution onto a solid support, thereby providing a solid support that binds to nucleic acids. Also provided herein is a method for treating a nucleic acid comprising exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acid onto the solid support, thereby providing a solid support that binds to the nucleic acid. Further provided herein are kits and compositions comprising such aqueous nucleic acid purification buffers, as well as related nucleic acid analysis devices, and uses of the kits.

Description

[0001] This invention relates to processes for preparing nucleic acids, and related buffers, kits, compositions, and apparatus. These methods may involve precipitating nucleic acids from a solution onto a solid support, and optionally, washing the solid support containing the nucleic acids and releasing the nucleic acids from the washed solid support. The invention also provides 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. Background Technology

[0002] The isolation of nucleic acids (such as DNA or RNA) is a crucial step in many molecular biology, medical, and diagnostic procedures. In fact, nucleic acids are often required to be isolated from complex mixtures in which they are frequently found before further research and procedures can be performed. For example, many types of nucleic acid detection, cloning, sequencing, amplification, hybridization, cDNA synthesis, and studies of nucleic acid structure and composition require relatively clean nucleic acid samples before related experiments can be conducted. Such complex mixtures often contain large amounts of cells or other contaminants (such as proteins or carbohydrates), which can hinder many reactions and techniques used in molecular biology. Furthermore, DNA can contaminate RNA preparations, and vice versa. Therefore, methods for isolating nucleic acids from complex mixtures (such as cells, tissues, etc.) are necessary not only from a preparative standpoint but also in many methods that rely on DNA or RNA identification, such as the diagnosis of microbial or viral infections, forensic medicine, tissue and blood typing, genotyping, and detection of genetic variations. Purifying DNA or RNA from relatively enriched but still contaminated samples is also desirable, for example, purifying synthetically prepared nucleic acid materials, such as purifying PCR products from contaminating salts, excess primers, and / or dNTPs.

[0003] Many methods for isolating nucleic acids are known, but these methods typically rely on a series of complex extraction and washing steps, making them time-consuming and labor-intensive. Furthermore, they often involve the use of materials such as alcohols and other organic solvents, dissociation agents, and proteases. This is disadvantageous because such materials tend to interfere with enzymatic reactions and other downstream processing applications.

[0004] In particular, typical methods for isolating nucleic acids from complex starting materials, such as blood or blood products or tissues, involve lysing the biological material with a detergent or dissociative agent (potentially in the presence of protein-degrading enzymes), followed by several extractions with organic solvents such as phenol and / or chloroform, ethanol precipitation, centrifugation, and nucleic acid dialysis. Purifying RNA from DNA may involve selective precipitation using LiCl, or selective separation using guanidine thiocyanate combined with phenol extraction and ethanol precipitation. These methods are not only cumbersome and time-consuming, but also require relatively many steps, increasing the risk of degradation, sample loss, or cross-contamination when processing multiple samples simultaneously. Furthermore, the reagents used in such purifications often have several other drawbacks, such as flammability, high viscosity, or potential environmental, health, or safety (EHS) issues.

[0005] Methods relying on the use of solid phases have been proposed. For example, US 5,234,809 describes a method in which nucleic acids are bound to a solid phase in the form of silica particles in the presence of a dissociating agent (such as a guanidine salt), thereby separating them from the remainder of the sample. WO 91 / 12079 describes a method for capturing nucleic acids on a solid phase surface by precipitation. Generally, a monohydric alcohol (ethanol or isopropanol) and a salt are used as precipitants.

[0006] When an aqueous medium contains sufficient amounts of ethanol and / or isopropanol, its role is to precipitate dissolved nucleic acids from the solution and retain nucleic acids already in the external phase. Therefore, ethanol and isopropanol can be described as “antisolventizing” agents for nucleic acids. Without being bound by any theory, it is believed that ethanol or isopropanol reduces the amount of free water available to dissolve nucleic acids. Therefore, these two monohydric alcohols are used in nucleic acid processing to precipitate them (e.g., bind them to a solid support) and / or prevent their dissolution in aqueous media, in the latter case, while other processes are carried out. In any given workflow, one or a combination of these alcohols can be used to precipitate nucleic acids, retain the nucleic acid precipitate, or simultaneously precipitate and retain the precipitate.

[0007] US 5,705,628 and US 5,898,071 describe methods for isolating nucleic acid fragments using a combination of high molecular weight polyalkylene glycols (e.g., polyethylene glycol) at concentrations of 7% to 13% with salts in the range of 0.5M to 5M to achieve binding to functional groups on a solid support used as a bioaffinity adsorbent for DNA. WO 2012 / 069660 A1 describes a method for separating nucleic acids onto a solid support using polyols. EP3215620B1 describes a method for extracting nucleic acids from plasma without a dissociation agent or volatile reagents.

[0008] While such methods generally accelerate the nucleic acid separation process, there are also some drawbacks to procedures that rely on existing reagents (such as alcohols (like ethanol or isopropanol), dissociation agents, salts, and high molecular weight molecules (such as polyethylene glycol)) to induce and / or maintain the solid-phase nucleic acid precipitation state.

[0009] Large molecular weight molecules increase liquid viscosity, which reduces the efficiency of purification schemes. In the case of magnetic bead separation, these large molecules slow down the separation rate because the contact time with the magnet must be increased to separate the beads. Furthermore, the presence of large molecular weight molecules makes it more difficult to remove the supernatant from such systems. Polyols perform better than large molecular weight molecules, but their viscosity is still higher than ideal, which can affect the accuracy of liquid transfer operations, especially in point-of-care (POC) or microfluidic devices with narrow channels, and may lead to reduced impurity removal efficiency.

[0010] The need for high molar concentrations of dissociative agents results in viscous solutions, which can be difficult to handle, especially in RNA work. Amplification procedures (such as PCR) and other enzyme-based reactions are highly sensitive to the inhibitory or interfering effects of alcohols and other reagents. Furthermore, the drying of nucleic acid precipitation that must follow alcohol (i.e., ethanol or isopropanol) precipitation, as well as issues with dissolving nucleic acids, are known to cause artifacts in enzyme-based processes such as amplification reactions. In addition, liquids with high ethanol or isopropanol content are volatile and flammable, increasing the costs of transporting and disposing of the liquid, and thus the overall cost of the process.

[0011] There is a need for improved nucleic acid isolation methods and improved reagents for such methods. EHS-friendly methods are particularly desirable, which are rapid and simple to operate, achieve good yields with minimal loss, and avoid (or at least reduce) the use of volatile solvents and ionizing agents or alcohol precipitation, or the use of high levels of salts and / or high-viscosity, high-molecular-weight compounds, and are preferably compatible with on-the-fly and near-on-the-fly applications. Summary of the Invention

[0012] This invention is based in part on the understanding that polar aprotic solvents exist that can be used as nucleic acid purification buffers, replacing previously used alcohols, polyols, polyethylene glycols, or similar compounds. This can provide numerous advantages, such as low viscosity for improved handling / ease of separation from any solid phase, and / or low flammability for improved safety, and / or good compatibility with downstream processes such as PCR amplification or mass spectrometry analysis.

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

[0014] The polar aprotic solvent may be present in an amount of at least about 2% by weight. The polar aprotic solvent may be present in an amount of no more than about 80% by weight. For example, the polar aprotic solvent may be present in an amount between about 4% by weight and about 75% by weight.

[0015] Polar aprotic solvents may have boiling points exceeding 100°C at standard atmospheric pressure. For example, polar aprotic solvents may have boiling points exceeding 150°C at standard atmospheric pressure.

[0016] 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 heteroatoms. A polar aprotic solvent may contain 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. A polar aprotic solvent may contain 2 O heteroatoms. A polar aprotic solvent may contain 3 O heteroatoms. A polar aprotic solvent may contain 1 O heteroatom and 1 or 2 N heteroatoms. O heteroatoms may include =O and / or -O-.

[0017] 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 example, 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.

[0018] At 20°C and standard atmospheric pressure, the viscosity of a polar aprotic solvent can be less than 50 cP. For example, at 20°C and standard atmospheric pressure, the viscosity of a polar aprotic solvent can be less than 40 cP or 30 cP; for example, at 20°C and standard atmospheric pressure, the viscosity of a polar aprotic solvent can be less than 25 cP.

[0019] Polar aprotic solvents may have a flash point of at least 50°C.

[0020] Polar aprotic solvents are liquids at 0°C and standard atmospheric pressure.

[0021] The polar aprotic solvent may be selected from one or more of the following: dihydro-L-glucoseenone, 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-glucoseenone, N-butylpyrrolidone-2-one, and dipropylene glycol dimethyl ether.

[0022] The purification buffer may further contain one or more of the following, or combinations thereof: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent. For example, the purification buffer may contain two or more of the following: a dissociation 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 dissociation 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, a purification buffer can be both a binding buffer and a washing buffer. A purification buffer can be a binding buffer. A purification buffer can be a washing buffer. The binding conditions for precipitating nucleic acids onto a solid support may be more stringent than the washing conditions that need to prevent the nucleic acids from eluting from the solid support.

[0026] Therefore, in some embodiments, the washing 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 washing buffer may contain 1.1 to 5 times the aqueous dilution of the binding buffer; for example, the washing buffer may contain 1.5 to 4 times the dilution of the binding buffer.

[0027] In an implementation, 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 different from the binding buffer and does not represent an aqueous dilution of the binding buffer.

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

[0029] A second aspect of the invention provides a purification buffer from the first aspect to precipitate nucleic acids from a solution onto a solid support, thereby providing a solid support for binding nucleic acids.

[0030] The solid support may contain surface hydroxyl functional groups. Surface hydroxyl functional groups may include silanol, carboxyl, or a portion of a sugar moiety.

[0031] Solid supports may include one or more of the following: particles, resin, beads, filters, boxes, columns, arrays, membranes, chips, disks, or glass slides. Solid supports may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports may be components of robotic liquid handling platforms.

[0032] This application may further include washing the solid support containing bound nucleic acids with a washing buffer. The washing buffer may be or contain a purification buffer. The binding conditions for precipitating nucleic acids onto the solid support may be more stringent than the washing conditions that need to prevent the nucleic acids from eluting from the solid support. Therefore, the washing buffer may represent an aqueous dilution of the purification buffer used for binding. For example, the washing buffer may contain a 1.1-fold to 5-fold dilution of the purification buffer; for example, the washing buffer may contain a 1.5-fold to 4-fold dilution of the purification buffer.

[0033] This application may further include contacting a solid support containing bound nucleic acids with an elution buffer to separate the nucleic acids from the solid support. The elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof.

[0034] The isolated nucleic acids can be subjected to one or more additional processes. These additional processes can be selected from detection, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, protein expression, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, drug or therapeutic agents, and genome editing.

[0035] Amplification can include PCR, qPCR, 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 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) (for an overview, see "..."). Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections (See Infection and Drug Resistance 2020:13, 455-483, and its references).

[0036] Sequencing can include next-generation sequencing. "Next-generation sequencing" and "high-throughput sequencing" are sequencing technologies that parallelize the sequencing process, generating 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), ligation sequencing (e.g., Life Technologies' SOLiD sequencing), synthetic sequencing 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 technologies (such as transmission electron microscopy).

[0037] Nucleic acids can be DNA. For example, DNA can be one or more of the following: synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g., dsDNA or ssDNA), cDNA, or cfDNA.

[0038] Nucleic acids can be RNA. 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, or viral RNA (e.g., dsRNA or ssRNA).

[0039] A third aspect provides a method for processing nucleic acids. The method includes: exposing a sample containing nucleic acids to an aqueous medium containing a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acids onto the solid support, thereby providing a solid support for binding nucleic acids.

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

[0041] The solid support may contain surface hydroxyl functional groups. Surface hydroxyl functional groups may include silanol, carboxyl, or a portion of a sugar moiety.

[0042] The solid support may include one or more of the following: particles, resin, beads, filters, boxes, columns, arrays, membranes, chips, disks, or glass slides. The solid support may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic.

[0043] The method may further include washing the solid support containing the bound nucleic acid with a washing buffer. The washing buffer may be or contain a purification buffer. The binding conditions for precipitating the nucleic acid onto the solid support may be more stringent than the washing conditions required to prevent the nucleic acid from eluting from the solid support. Therefore, the washing buffer may represent an aqueous dilution of the purification buffer used for binding. For example, the washing buffer may contain a 1.1-fold to 5-fold dilution of the purification buffer; for example, the washing buffer may contain a 1.5-fold to 4-fold dilution of the purification buffer.

[0044] The method may further include contacting the solid support containing the bound nucleic acid with an elution buffer to separate the nucleic acid from the solid support, thereby providing isolated nucleic acid. The elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof.

[0045] The method may further include subjecting nucleic acids (e.g., isolated nucleic acids) to one or more additional processes. These additional processes may be selected from detection, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, protein expression, hybridization, blotting, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic agents, and genome editing.

[0046] Amplification can include PCR, qPCR, 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 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) (for an overview, see "..."). Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections (See Infection and Drug Resistance 2020:13, 455-483, and its references).

[0047] Sequencing can include next-generation sequencing. "Next-generation sequencing" and "high-throughput sequencing" are sequencing technologies that parallelize the sequencing process, generating 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), ligation sequencing (e.g., Life Technologies' SOLiD sequencing), synthetic sequencing 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 technologies (such as transmission electron microscopy).

[0048] Samples may include pretreated or untreated biological samples, clinical environmental samples, or enzyme reaction mixtures. Samples may include pretreated or untreated biological samples. Biological samples may be present in physiological buffers or transport media. Biological samples may be collected or biopsied samples or cultured samples. Samples may include environmental samples. Samples may include enzyme reaction mixtures.

[0049] Biological samples can be any suitable biological sample. Exemplary biological samples may include one or more of the following: blood, bloodstains, umbilical cord blood, blood components (e.g., platelet concentrate), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, erythrocyte sedimentation rate (ESR) leukocytes, serum, plasma, saliva, saliva stains, oral cells, oral swabs, semen, semen stains, urine, feces, fecal stains, cigarette butts, chewing gum, formalin-fixed paraffin-embedded (FFPE) samples, 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, exhalation, 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. In some cases, biological samples can be clinical samples. In other cases, samples can be cell-free samples.

[0050] Environmental samples may include water samples (such as wastewater samples), soil samples, sediment samples, surface swabs, air-derived samples (such as air filter residues), cosmetics, food ingredients or food samples, or combinations thereof.

[0051] The enzyme reaction mixture can be any such mixture that may contain nucleic acid sequences. 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 reaction mixture, a nucleic acid assembly reaction mixture, or a barcode reaction mixture.

[0052] The process of this invention can be used to extract and analyze fragmented DNA. DNA can be fragmented by a variety of methods known in the art, such as nuclease digestion (including digestion using restriction endonucleases and DNases), sonication, heating, mechanical disruption (such as by shearing or vortexing), and chemical treatment. Suitable chemical treatments include, for example, the use of metal ions, such as iron (Zhang et al., Nucl. Acids Res. 29(13):e66, 2001), oxidizing agents, such as bisulfite (Ehrich et al., Nucl. Acids Res. 35(5):e29, 2007), and antibiotics and drugs, such as bleomycin (Chen et al., Nucl. Acids Res. 3<5(11):3781-3790, 2008). The preparation of fragmented DNA may contain fragments of a range of sizes, or the size range may be relatively limited. Those skilled in the art will recognize that the actual size of the fragment will be determined by factors such as the fragmentation method chosen and the conditions used (e.g., processing time).

[0053] In this embodiment, the aqueous medium does not contain ethanol, isopropanol, or 2-methyl-1,3-propanediol. In this embodiment, the aqueous medium does not contain dimethyl sulfoxide (DMSO). In this embodiment, the aqueous medium does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, or DMSO. In this embodiment, the aqueous medium does not contain alcohols (including any glycols or polyols). In this embodiment, the aqueous medium does not contain alcohols (including any glycols or polyols) or DMSO. In this embodiment, the aqueous medium does not contain salts or liquid release agents.

[0054] The method may further include performing point-of-care testing. Point-of-care testing may include pathogen detection and / or biological warfare agent detection 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.

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

[0056] The solid support may contain surface hydroxyl functional groups. Surface hydroxyl functional groups may include silanol, carboxyl, or a portion of a sugar moiety.

[0057] Solid supports may include one or more of the following: particles, resin, beads, filters, boxes, columns, arrays, membranes, chips, disks, or glass slides. Solid supports may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic. In some cases, solid supports may be components of robotic liquid handling platforms.

[0058] The kit may include 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.

[0059] A fifth aspect provides a material composition. The material composition comprises (i) a solution containing an aqueous nucleic acid purification buffer according to the first aspect; and (ii) a nucleic acid solid support.

[0060] The solid support may contain surface hydroxyl functional groups. Surface hydroxyl functional groups may include silanol, carboxyl, or a portion of a sugar moiety.

[0061] The solid support may include one or more of the following: particles, resin, beads, filters, boxes, columns, arrays, membranes, chips, disks, or glass slides. The solid support may include beads, such as monodisperse beads. (Optionally, monodisperse) beads may be magnetic.

[0062] Nucleic acids can be DNA; such as plasmid DNA, cDNA, cfDNA, and / or genomic DNA. Nucleic acids can also be RNA; such as one or more of the following: mRNA (e.g., isolated from biological samples or...). in vitro Transcribed RNA, siRNA, microRNA, tRNA, or cfRNA.

[0063] The sixth aspect provides the use of the kits from the fourth aspect in automated nucleic acid analysis platforms.

[0064] The seventh aspect provides a nucleic acid analysis device. This device includes an automated nucleic acid analysis platform and a reagent kit as described in the fourth aspect. The automated nucleic acid analysis platform includes a portion configured to contain a solid support.

[0065] Automated nucleic acid analysis platforms can be point-of-care testing (POC) instruments. POC instruments can be used for pathogen detection and / or biological warfare agent detection, and / or genetic disease detection. Pathogen detection or biological warfare agent detection can include nucleic acid detection of viruses, bacteria, single-celled fungi, or protozoa.

[0066] This invention has a wide range of applications in laboratory research, human and veterinary medicine, public health and environmental hygiene, 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 fields requiring the preparation and analysis of DNA from biological samples. Attached Figure Description

[0067] The embodiments of the present invention are further described below with reference to the accompanying drawings, wherein: Figure 1 provides an overview of an exemplary workflow for isolating nucleic acids from a liquid sample. (A) provides a general schematic diagram, (B) provides an example of a standard workflow for isolating nucleic acids using isopropanol (IPA) in a binding buffer, and (C) provides an exemplary workflow of the present invention using N-butylpyrrolidone-2-one (TamiSolve™) in a binding buffer. As those skilled in the art will understand, the exemplary workflow (C) can be adapted to use other binding buffers and washing buffers as disclosed herein.

[0068] Figure 2 shows the DynaMag™-2 magnet, SKU 12321D.

[0069] Figure 3 illustrates the recovery of nucleic acids from a silanol-functionalized stationary phase using aprotic solvents selected from the binding buffer, namely dihydro-L-glucoselenone (Cyrene™) and dipropylene glycol dimethyl ether (Proglyde™). N -Butylpyrrolidone-2-one (TamiSolve™) or N-formylmorpholine (N-FM), with the proton solvent isopropanol (IPA) used as a positive control. (A) shows the MS2 RNA recovery rate on silane beads, while (B) shows the M13 DNA recovery rate on silane beads.

[0070] Figure 4 illustrates the recovery of nucleic acids from a carboxylic acid-functionalized stationary phase using a nonprotic solvent selected from Cyrene™, Proglyde™, TamiSolve™, or N-formylmorpholine (N-FM) in binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. (A) shows the recovery rate of MS2 RNA on carboxylic acid beads, while (B) shows the recovery rate of M13 DNA on carboxylic acid beads.

[0071] Figure 5 shows the PCR results of eight different extractions of nucleic acids recovered from the carboxylic acid-functionalized stationary phase, including a completely aprotic solvent workflow. (A) shows the results for RNA. (B) shows the results for DNA.

[0072] Figure 6 shows the percentage recovery calculated based on the copy number of nucleic acids incorporated into the sample. (A) shows the results for RNA. (B) shows the results for DNA. Detailed Implementation

[0073] Throughout the description and claims of this specification, the terms "comprise" and "contain," and variations thereof, mean "including (but not limited to)," and are not intended to exclude other parts, additives, components, integers, or steps. Throughout this specification and claims, the singular form includes the plural form unless the context otherwise requires. In particular, where the indefinite article is used, the specification should be understood to consider both the plural and singular forms unless the context otherwise requires.

[0074] Features, integers, properties, compounds, chemical parts, or groups described in connection with a particular aspect, embodiment, or example 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 may 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 any novel step or any novel combination of steps of any method or process so disclosed.

[0075] Readers should note all papers and documents related to this application that were submitted concurrently with or prior to this specification and that are open to the public for review of this application, the contents of which are incorporated herein by reference.

[0076] 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.

[0077] 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.

[0078] definition

[0079] The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in practicing this disclosure.

[0080] 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., "hybridization" or the formation of a "double strand". Thus, nucleic acids can be DNA or RNA or any modification thereof, including conformationally restricted or nucleobase analogs 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 exists in a separate strand or in the same strand, and 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. Nucleic acids used in various embodiments may comprise nucleotides or combinations thereof in the form of chemical, enzymatic, or metabolic modifications, 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).

[0081] 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 or precipitated onto a solid support. Therefore, an exemplary purification buffer can also be considered a binding buffer or a washing buffer.

[0082] 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 may 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.

[0083] 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 washing buffers, as they should minimize the loss of nucleic acids from the solid support during washing. Binding conditions for precipitating nucleic acids onto a solid support may be more stringent than washing conditions that require preventing the nucleic acids from eluting from the solid support. Therefore, a washing buffer can represent an aqueous dilution of the binding buffer, such as a 1.1 to 5-fold dilution of the corresponding binding buffer.

[0084] 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 are 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 thin-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 a solid support includes beads, monodisperse (magnetic or non-magnetic) beads are preferred because monodisperse beads can provide more consistent performance in assays. Solid supports may contain magnetic beads. The solid support used in practicing this invention has a hydrophilic surface that allows nucleic acids to bind, for example, through non-covalent interactions. The hydrophilic surface of the solid support may include a charged surface in a pH range of about 6 to about 8.

[0085] Unless otherwise stated, the term "nucleic acid solid support" means a solid support or solid phase as disclosed herein suitable for binding nucleic acids (e.g., through non-covalent interactions). Nucleic acid solid supports may have hydrophilic surfaces that allow nucleic acid binding. The hydrophilic surfaces of solid supports may include charged surfaces in a pH range of about 6 to about 8.

[0086] 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 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 a 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 "mean" is the average particle size or bead diameter 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, the proportion of monodisperse particle or bead groups whose size is within ±5% of the average diameter may exceed 90%, preferably more than 95%.

[0087] 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, as well as the surfaces of the solvent-accessible pores of a porous solid support. The surface of a solid support may contain surface hydroxyl groups. These surface hydroxyl groups may comprise 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 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 The solid support may contain ionizable positively charged 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 6914137 B2, and covalently bound as described in column 7, lines 29 through 64 of the same document, the entire contents of which are incorporated herein by reference.

[0088] 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.

[0089] 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 can 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.

[0090] 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, its magnetism shuts off when the magnetic field is removed. When a magnetic material is superparamagnetic, it becomes saturated under relatively low magnetic fields, and its magnetism shuts 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., less than about 15 nanometers for iron oxide).

[0091] The term "flash point" for a liquid refers to the lowest temperature at which it can be ignited in its liquid or vapor form. For example, a solvent with a flash point of 2 degrees Celsius is easily ignited in almost any normal workplace and therefore requires precautions, while solvents with flash points above 50 degrees Celsius only pose a risk of ignition under specific extreme conditions. The table below lists the flash points of some common solvents.

[0092]

[0093] buffer solution

[0094] Methods for processing nucleic acids typically involve exposing the nucleic acids to an aqueous solution and then precipitating them 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.

[0095] This article provides buffer solutions that can be used for nucleic acid preparation, particularly for precipitating nucleic acids from solution onto solid supports and / or for washing solid supports bound to nucleic acids.

[0096] One aspect provides an aqueous nucleic acid purification buffer containing a polar aprotic solvent. These aqueous nucleic acid purification buffers offer numerous advantages compared to solvents used in the prior art, such as alcohols and polyols. For example, polar aprotic solvents are generally non-flammable and have low 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 sealed containers and boxes for immediate use and transported. Low volatility also reduces component concentration over time. Polar aprotic solvents typically have relatively low viscosity (e.g., less than 50 cP at 20°C and standard atmospheric pressure), which allows for good levels of accuracy and ease of measurement and dispensing of the buffer. The buffer also offers other benefits such as relative environmental friendliness (including biodegradable and renewable components), compliance with health and safety standards, and compatibility with downstream nucleic acid processing / analysis procedures such as amplification or mass spectrometry.

[0097] The polar aprotic solvent may be present in an amount of at least about 2% by weight. The polar aprotic solvent may be present in an amount of no more than about 80% by weight. For example, the polar aprotic solvent may be present in an amount between about 4% by weight and about 75% by weight.

[0098] Polar aprotic solvents may have boiling points exceeding approximately 100°C at standard atmospheric pressure. For example, polar aprotic solvents may have boiling points exceeding approximately 150°C at standard atmospheric pressure.

[0099] 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 heteroatoms. A polar aprotic solvent may contain 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. A polar aprotic solvent may contain 2 O heteroatoms. A polar aprotic solvent may contain 3 O heteroatoms. A polar aprotic solvent may contain 1 O heteroatom and 1 or 2 N heteroatoms. O heteroatoms may include =O and / or -O-.

[0100] Polar aprotic solvents may consist of carbon atoms (e.g., 4, 5, 6, 7, 8, 9, or 10 carbon atoms; e.g., 6, 7, 8, or 9 carbon atoms), hydrogen atoms, and 2, 3, or 4 heteroatoms selected from O and N. For example, 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 heteroatoms. A polar aprotic solvent may contain 1 or 2 O heteroatoms and 1 or 2 N heteroatoms. A polar aprotic solvent may contain 2 O heteroatoms. A polar aprotic solvent may contain 3 O heteroatoms. A polar aprotic solvent may contain 1 O heteroatom and 1 or 2 N heteroatoms. O heteroatoms may include =O and / or -O-.

[0101] Polar aprotic solvents may contain 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, a polar aprotic solvent may contain 6, 7, 8, 9, or 10 carbon atoms; for example, a polar aprotic solvent may contain 6, 7, or 8 carbon atoms. For example, a polar aprotic solvent may contain 5, 6, 7, or 8 carbon atoms; for example, a polar aprotic solvent may contain 6 or 8 carbon atoms. A polar aprotic solvent may contain 5 carbon atoms. A polar aprotic solvent may contain 6 carbon atoms. A polar aprotic solvent may contain 7 carbon atoms. A polar aprotic solvent may contain 8 carbon atoms.

[0102] The viscosity of polar aprotic solvents at 20°C and standard atmospheric pressure may be less than about 50 cP. For example, the viscosity of polar aprotic solvents at 20°C and standard atmospheric pressure may be less than about 40 cP or about 30 cP; for example, the viscosity of polar aprotic solvents at 20°C and standard atmospheric pressure may be less than about 25 cP. Viscosity can be measured according to standard methods, such as those described in OECD (2012). Test No. 114: Liquid Viscosity OECD Guide to Chemical Testing, Chapter 1, OECD Press, Paris, https: / / doi.org / 10.1787 / 9789264185180-en, the contents of which are incorporated herein by reference.

[0103] Polar aprotic solvents may have a flash point of at least about 50°C.

[0104] Polar aprotic solvents are liquid at about 0°C and standard atmospheric pressure.

[0105] The polar aprotic solvent may be selected from one or more of the following: dihydro-L-glucoseenone, 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-glucoseenone, N-butylpyrrolidone-2-one, and dipropylene glycol dimethyl ether.

[0106] Polar aprotic solvents may be or contain N-butylpyrrolidone-2-one (Tamisolve™): .

[0107] Polar aprotic solvents may be or contain dihydro-L-glucoseenone (Cyrene™): .

[0108] Polar aprotic solvents may be or contain dipropylene glycol dimethyl ether (Proglyde™): .

[0109] Purification buffers may further contain one or more of the following, or combinations thereof: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent. For example, a purification buffer may contain two or more of the following: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent; or, for example, a purification buffer may contain three or more of the following: a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent.

[0110] Exemplary dissociation agents include guanidine salts (such as GuSCN and GuHCl), urea, etc. Surfactants can be selected from nonionic surfactants (such as Triton™ X-100, DDM, digitoxin, Tween...). ® 20. Tween ® 80. Ecosurf™, etc.), anionic surfactants (such as sodium lauryl sulfate, deoxycholate, cholate, sodium lauryl creatine, etc.), and zwitterionic surfactants (such as CHAPS, Zwittergent, etc.). ®(3-14, etc.). The buffer 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, trimethylglycine, TAPS, PBS, acetate, borate, HEPES, diglycine, MOPS, CHES, carbonate, etc., and mixtures thereof; the buffer may also contain other components such as polyethylene glycol (PEG), tetraethylene glycol (TEG), and linear polyacrylamide (LPA). The enzyme is typically a protein or other degrading enzyme, such as proteinase K or lysozyme. Exemplary inorganic salts include metal halides such as MgCl2, NaCl, LiCl, etc. However, in some embodiments, the purification buffer may not contain salts and / or a dissociating agent.

[0111] The purification buffer may further contain moderate amounts (e.g., less than about 20%, less than about 10%, or less than about 5%) of alcohol, polyol, polyethylene glycol (PEG), tetraethylene glycol (TEG), or linear polyacrylamide (LPA), or combinations thereof. The purification buffer may contain moderate amounts (e.g., less than about 20%, less than about 10%, or less than about 5%) of alcohol or polyol. The purification buffer may contain moderate amounts (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 having no more than moderate amounts (e.g., less than about 5%, or less than about 2%) of alcohol and / or polyol and / or PEG, and / or TEG and / or LPA is preferred. A purification buffer having no more than moderate amounts (e.g., less than about 5%, or less than about 2%) of alcohol or polyol is preferred.

[0112] Purification buffers can be binding buffers and / or washing buffers. For example, a purification buffer can be both a binding buffer and a washing buffer. A purification buffer can be a binding buffer. A purification buffer can be a washing buffer. Binding conditions for precipitating nucleic acids onto a solid support may be more stringent than washing conditions that merely aim to prevent (or at least minimize) the elution of nucleic acids from the solid support.

[0113] Therefore, a 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. A wash buffer may contain 1.1 to 5 times the dilution of the binding buffer; for example, a wash buffer may contain 1.5 to 4 times the dilution of the binding buffer.

[0114] Therefore, a workflow or kit may include both the binding buffer and the washing buffer of the present invention. In some embodiments, the washing buffer may be the same as the binding buffer (or an aqueous dilution of the binding buffer). In other embodiments, 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.

[0115] In this embodiment, the purification buffer does not contain ethanol, isopropanol, or 2-methyl-1,3-propanediol. In this embodiment, the purification buffer does not contain dimethyl sulfoxide (DMSO). In this embodiment, the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, or DMSO. In this embodiment, the purification buffer does not contain alcohols (including any diols or polyols). In this embodiment, the purification buffer does not contain alcohols (including any diols or polyols) or DMSO.

[0116] Methods and uses

[0117] Figure 1A provides a schematic diagram illustrating the general process for nucleic acid preparation. In the first step, a liquid sample containing nucleic acids is provided. A solid support is added to the liquid sample, followed by a purification buffer (such as a binding buffer). The nucleic acids are allowed to precipitate from the solution onto the solid support to provide a solid phase containing bound nucleic acids. The liquid is separated from the bound solid support, and the solid support is washed once or multiple times with a washing buffer to provide a cleaned solid phase containing bound nucleic acids. Optionally, the cleaned solid phase containing bound nucleic acids is dried to provide a dried solid phase containing bound nucleic acids. An elution buffer can be added to either the cleaned or dried solid phase containing bound nucleic acids to provide eluted nucleic acids. The eluted nucleic acids can be used for downstream nucleic acid processing / analysis. Furthermore, for some downstream applications, the elution step is not necessary; that is, either the cleaned or dried solid phase containing bound nucleic acids can be used for downstream nucleic acid processing / analysis.

[0118] Figure 1B illustrates a standard workflow using a purification buffer / binding buffer containing isopropanol (IPA), a wash buffer 1 containing 50% lysis / binding buffer (LBB) and 50% IPA, and a wash buffer 2 containing 70% ethanol. Figure 1C illustrates an exemplary workflow of the present invention using a purification buffer / binding buffer containing TamiSolve™, a wash buffer 1 containing 50% lysis / binding buffer (LBB) and 50% TamiSolve™, and a wash buffer 2 containing either 50% TamiSolve™ or 50% 2-butoxyethanol (2-BE). The workflow in Figure 1C can also be performed using other exemplary polar aprotic solvents as disclosed herein, such as dihydro-L-glucoselenone, dipropylene glycol dimethyl ether, and N-formylmorpholine, instead of TamiSolve™.

[0119] Prior to the general process shown in Figure 1A, nucleic acids can be initially extracted from the sample, for example, through Figure 1B and Figure 1 The lysis step is shown in C. This lysis and binding of the nucleic acids released onto the solid support can be performed using the same lysis / binding buffer used from the lysis step to the binding step.

[0120] 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, dissociative agents in lysis solutions can reduce enzyme activity and promote induced cell lysis. Any suitable dissociative agent can be used in the lysis or lysis / binding buffer. For example, in some embodiments, the dissociative agent is selected from guanidine salts (e.g., guanidine isothiocyanate or guanidine chloride), lithium perchlorate, lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and combinations thereof. The dissociative agent (chaotropic agent or chaotrope) may be present in the lysis or lysis / binding buffer at a concentration sufficient to denature macromolecules in biological materials 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 2M, at least about 3M, such as at least about 3.5M or about 4.0M, for example, about 3M to 6M. In a preferred embodiment, the lysis buffer contains guanidine isothiocyanate at a concentration of about 3.5 M to 4.5 M, for example, about 4.0 M.

[0121] Lysis or lysis / binding buffers typically contain surfactants, such as nonionic surfactants or detergents, among which polyethylene oxide-containing surfactants may be mentioned. Detergents in the lysis solution are used to disrupt cell and organelle membranes (e.g., lyse cells and organelles) and denature proteins in the biological material. Therefore, detergents are used to facilitate the release of nucleic acids from cells and other entities (e.g., viruses) in the biological material. Any suitable detergent, such as a nonionic detergent, can be used in the lysis or lysis / binding solution. For example, in some embodiments, the detergent is selected from sodium lauroyl sarcosinate (sarkosyl), sodium dodecyl sulfate (SOS), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), and combinations thereof. In a preferred embodiment, the detergent is selected from sodium lauroyl sarcosinate (sarkosyl) and 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100). The detergent concentration in the lysis solution is sufficient to disrupt cell membranes and organelle membranes, such as lysing cells and organelles, disrupting viral envelopes and / or capsids, and / or denaturing proteins in biological material. In some embodiments, the detergent is present at concentrations of 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% w / v. In some preferred embodiments, the detergent is sodium lauroyl sarcosinate (sarkosyl) or 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100) at concentrations as defined above.

[0122] The lysis / binding buffer can be any buffer known for this purpose, such as a buffer with a high concentration of a dissociating agent (e.g., guanidine salt or urea) and a surfactant, such as Triton X-100. For example, a preferred lysis buffer is a buffer™ containing a high concentration of detergent and guanidine isothiocyanate (GTC).

[0123] 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. The role of such chelating agents is to isolate divalent cations, which is essential for enzymes that act on nucleic acids, such as DNases and RNases. Therefore, the role of chelating agents 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 concentration of the chelating agent may be sufficient to inhibit nucleic acid-degrading enzymes (i.e., in samples containing biological material) present in the lysis solution, for example, 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.

[0124] Lysis or lysis / binding buffers may also contain one or more reducing agents. When a reducing agent is present in the lysis buffer, it is used to reduce the disulfide bonds of proteins in the 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 may be 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.

[0125] When a reducing agent is included in the lysis or lysis / binding buffer, the reducing agent is present at a concentration sufficient to reduce the disulfide bonds of proteins in the biological material. In some embodiments, the reducing agent may be present at concentrations 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 reducing agent may 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.

[0126] 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 can promote nucleic acid aggregation and adsorption to a solid support (e.g., silica-coated magnetic beads) induced by a precipitant. Nucleic acid carriers are typically polymers, such as nucleic acids or polysaccharides. For example, nucleic acid carriers 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 embodiments, 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.

[0127] When a nucleic acid carrier is contained in a lysis or lysis / binding buffer, the nucleic acid carrier is present at a concentration sufficient to increase the efficiency of nucleic acid recovery from biological material (i.e., sufficient to increase the adsorption of nucleic acid on a solid support). In some embodiments, the nucleic acid carrier may be present in a 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.

[0128] 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., an endopeptide, such as proteinase K). In some embodiments, the lysis buffer may contain a protease (e.g., an endopeptide). Alternatively, in some embodiments, the protease (e.g., an endopeptide) 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., an endopeptide) may be added simultaneously with the sample contacting a solid support (e.g., a suspension of magnetic particles).

[0129] 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 ethanol, to facilitate nucleic acid capture on a solid support. However, this is not ideal, for example, because isopropanol is volatile and flammable.

[0130] One aspect of the invention provides a purification buffer for precipitating nucleic acids from solution onto a solid support, thereby providing a solid support for binding nucleic acids.

[0131] The solid support may contain surface hydroxyl functional groups. These surface hydroxyl functional groups may comprise a portion of a silanol, carboxyl, or sugar moiety. The surface hydroxyl functional groups may comprise a portion of a silanol moiety. The surface hydroxyl functional groups may comprise a portion of a carboxyl moiety. The surface hydroxyl functional groups may comprise a portion of a sugar moiety. The sugar moiety may be or may comprise one or more polysaccharides, such as complex branched dextran, like dextran.

[0132] Solid supports may include one or more of the following: particles, resin, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. 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.

[0133] In some cases, magnetic beads may contain microparticles or nanoparticles. In some examples, magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application number GB2210796.5, which is incorporated herein by reference, can be used. In some embodiments, the solid support may be selected from any commercially available solid support suitable for binding nucleic acids. For example, solid supports may be selected from Dynabeads™ MyOne™ Silane, Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M-270™ Carboxylic Acid, Dynabeads™ Oligo(dT)25 magnetic beads (all from Thermo Fisher Scientific), SeraMag SpeedBeads™ carboxylate-modified or SeraSil-Mag 400 or 700 (Cytiva), BioMagPlus COOH™ and ProMag 1 COOH™ (all from Bangs Laboratories, INCFishers), 4.4 µm fluorescent ferromagnetic beads or 2.0 µm ferromagnetic beads (all from Spherotech INC, Lake Forest, Illinois), 2 µm beads named WHM-S001™ or 2 µm beads named WHM-S002™ (both from Creative). Diagnostics, New York, NY; silanol magnetic microspheres or carboxyl magnetic microspheres or oligo(dT) magnetic microspheres (available in various nm or µm sizes from VDO Biotech, Suzhou, China); carboxyl Adembeads (available in 100 nm, 200 nm, 300 nm or 500 nm sizes) or carboxyl mother beads (500 nm) (from Ademtech, France); MagneSil™ beads (from Promega); BeaverBeads™ Mag COOH (from Beaver Biomedical Engineering Ltd).LodeStars High Bind Carboxyl beads (from Agilent), Magnosphere™, MS300 Carboxyl, MS 160 Carboxyl, or MS160 Carboxyl (all from JSR Life Sciences), PureProteome Carboxy FlexiBind bead system (available in various bead sizes from Sigma-Aldrich), BioMag™ Carboxyl, BioMag™ Maxi Carboxyl, or BioMag™ Plus Carboxyl (all from Polysciences), Carboxyl Super Mag or Mono Mag magnetic beads (available in sizes between 0.1 µm and 4.5 µm from Ocean Nanotech), and Carboxyl magnetic beads (available in various sizes from VdoBiotech).

[0134] This application may further include washing the solid support containing bound nucleic acids with a washing buffer. The washing buffer may be or contain a purification buffer; optionally, it may be mixed with other components. The binding conditions for precipitating nucleic acids onto the solid support may be more stringent than the washing conditions required to prevent the nucleic acids from eluting from the solid support. Therefore, the washing buffer may represent an aqueous dilution of the purification buffer used for binding. For example, the washing buffer may contain a 1.1-fold to 5-fold dilution of the purification buffer; for example, the washing buffer may contain a 1.5-fold to 4-fold dilution of the purification buffer.

[0135] This application can further include contacting a solid support containing bound nucleic acids with an elution buffer to separate the nucleic acids from the solid support. The elution buffer may contain water, Tris-HCl, EDTA, or a combination thereof. Conventional elution buffers, such as aqueous Tris buffers with a pH greater than 7, such as 10 Mm Tris-HCl buffer at pH 8.0, can be used.

[0136] The isolated nucleic acids may be subjected to one or more additional processes. These additional processes may be performed when the isolated nucleic acids are bound to a solid support (optionally after washing the solid support to which the nucleic acids are bound), or after they have been separated from the solid support.

[0137] One or more additional processes may be selected from detection, cloning, restriction enzyme digestion, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, protein expression, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, drug or therapeutic preparations, and genome editing.

[0138] Amplification can include PCR, qPCR, reverse transcription, in vitro Transcription or isothermal amplification. Isothermal amplification may include 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) (for an overview, see "..."). Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections (See Infection and Drug Resistance 2020:13, 455-483, and its references).

[0139] Sequencing can include next-generation sequencing. "Next-generation sequencing" and "high-throughput sequencing" are sequencing technologies that parallelize the sequencing process, generating 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), ligation sequencing (e.g., Life Technologies' SOLiD sequencing), synthetic sequencing 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 technologies (such as transmission electron microscopy).

[0140] Nucleic acids can be DNA. For example, DNA can be one or more of the following: synthetic DNA (e.g., DNA assembled from synthetic oligonucleotides, enzymatically synthesized DNA, doggy-bone DNA, dumbbell DNA, etc.), plasmid DNA, genomic DNA, viral DNA (e.g., dsDNA or ssDNA), cDNA, or cfDNA.

[0141] Nucleic acids can be RNA. 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, or viral RNA (e.g., dsRNA or ssRNA).

[0142] Another approach provides a method for processing nucleic acids. This method includes: exposing a sample containing nucleic acids to an aqueous medium containing a polar aprotic solvent in the presence of a solid support; and precipitating the nucleic acids onto the solid support, thereby providing a solid support for binding nucleic acids.

[0143] In a preferred embodiment, the aqueous medium containing a polar aprotic solvent is an aqueous nucleic acid purification buffer as defined in the first aspect.

[0144] The solid support may contain surface hydroxyl functional groups. These surface hydroxyl functional groups may comprise a portion of a silanol, carboxyl, or sugar moiety. The surface hydroxyl functional groups may comprise a portion of a silanol moiety. The surface hydroxyl functional groups may comprise a portion of a carboxyl moiety. The surface hydroxyl functional groups may comprise a portion of a sugar moiety. The sugar moiety may be or may comprise one or more polysaccharides, such as complex branched dextran, like dextran.

[0145] Solid supports may include one or more of the following: particles, resin, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. 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. (Optionally monodisperse) Beads may be magnetic.

[0146] The method may further include washing the solid support containing the bound nucleic acid with a washing buffer. The washing buffer may be or contain a purification buffer. The binding conditions for precipitating the nucleic acid onto the solid support may be more stringent than the washing conditions required to prevent the nucleic acid from eluting from the solid support. Therefore, the washing buffer may represent an aqueous dilution of the purification buffer used for binding. For example, the washing buffer may contain a 1.1-fold to 5-fold dilution of the purification buffer; for example, the washing buffer may contain a 1.5-fold to 4-fold dilution of the purification buffer.

[0147] The method may further include contacting the solid support containing the bound nucleic acid with an elution buffer to separate the nucleic acid from the solid support, thereby providing isolated nucleic acid. 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.

[0148] The method may further include subjecting nucleic acids (e.g., isolated nucleic acids) to one or more additional processes. These additional processes may be performed when the isolated nucleic acids are bound to a solid support (optionally after washing the solid support to which the nucleic acids are bound), or after they have been separated from the solid support.

[0149] One or more additional processes may be 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, drug or therapeutic preparations, and genome editing.

[0150] Amplification can include PCR, qPCR, reverse transcription, in vitro Transcription or isothermal amplification. Isothermal amplification may include 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) (for an overview, see "..."). Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections (See Infection and Drug Resistance 2020:13, 455-483, and its references).

[0151] Sequencing can include next-generation sequencing. "Next-generation sequencing" and "high-throughput sequencing" are sequencing technologies that parallelize the sequencing process, generating 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), ligation sequencing (e.g., Life Technologies' SOLiD sequencing), synthetic sequencing 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 technologies (such as transmission electron microscopy).

[0152] Samples may include pretreated or untreated biological samples, environmental samples, or enzyme reaction mixtures. Samples may include pretreated or untreated biological samples. Biological samples may be present in physiological buffers or transport media. Samples may include environmental samples. Samples may include enzyme reaction mixtures.

[0153] Biological samples can be any suitable biological sample. Exemplary biological samples may include one or more of the following: blood, bloodstains, umbilical cord blood, blood components (e.g., platelet concentrate), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, erythrocyte sedimentation rate (ESR) leukocytes, serum, plasma, saliva, saliva stains, oral cells, oral swabs, semen, semen stains, urine, feces, fecal stains, cigarette butts, chewing gum, formalin-fixed paraffin-embedded (FFPE) samples, 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, 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. In some cases, biological samples can be clinical samples. In other cases, samples can be cell-free samples.

[0154] Environmental samples may include water samples (such as wastewater samples), soil samples, sediment samples, surface swabs, air-derived samples (such as air filter residues), cosmetics, food ingredients or food samples, or combinations thereof.

[0155] The enzyme reaction mixture can be any such mixture that may contain nucleic acid sequences. 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, an amplification reaction mixture, a library preparation reaction mixture, a restriction reaction mixture, a nucleic acid assembly reaction mixture, or a barcode reaction mixture.

[0156] In this embodiment, the aqueous medium does not contain ethanol, isopropanol, or 2-methyl-1,3-propanediol. In this embodiment, the aqueous medium does not contain dimethyl sulfoxide (DMSO). In this embodiment, the aqueous medium does not contain ethanol, isopropanol, 2-methyl-1,3-propanediol, or DMSO. In this embodiment, the aqueous medium does not contain alcohols (including any glycols or polyols). In this embodiment, the aqueous medium does not contain alcohols (including any glycols or polyols) or DMSO.

[0157] This method may further include point-of-care (POC) testing. POC testing may include pathogen detection and / or biological warfare agent detection and / or genetic disease detection, and / or may be used for genetic screening. Pathogen detection or biological warfare agent detection may include nucleic acid detection of viruses, bacteria, single-celled fungi, or protozoa.

[0158] 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 washing 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 it is advantageous if the reagents contained therein (such as an aqueous medium containing a polar aprotic solvent, and / or a buffer) are non-flammable.

[0159] 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: Samples containing nucleic acids are exposed to an aqueous medium containing a polar aprotic solvent in the presence of a solid support. Nucleic acid is precipitated onto a solid support, thereby providing a solid support for binding nucleic acid; The solid support containing bound nucleic acids was washed with washing buffer; and The solid support containing bound nucleic acids is contacted with elution buffer to separate the nucleic acids from the solid support, thereby providing the separated nucleic acids; Then, one or more additional processes are performed on the nucleic acid. Examples of additional processes that may be associated with point-of-care testing include detection, cloning, analysis, epigenetic analysis, sequencing, amplification, research, transfection, protein expression, hybridization, cDNA synthesis, size separation, chromatography, and mass spectrometry.

[0160] Examples of instantaneous instruments suitable for use with 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.

[0161] Reagent kits, compositions and devices

[0162] One important advantage of this invention is that the polar aprotic solvent used in this invention can be used in harmless buffer solutions and is therefore ideal for packaging and transporting in kits.

[0163] Therefore, one aspect of the present invention is a kit comprising the aqueous nucleic acid purification buffer of the present invention (e.g., the first aspect) and a solid support.

[0164] Solid supports may include one or more of the following: particles, resins, beads, filters, boxes, columns, arrays, membranes, chips, disks, or glass slides. Solid supports may contain surface hydroxyl functional groups. Surface hydroxyl functional groups may contain a portion of a silanol, carboxyl, or sugar moiety. Surface hydroxyl functional groups may contain a portion of a silanol moiety. Surface hydroxyl functional groups may contain a portion of a carboxyl moiety. Surface hydroxyl functional groups may contain a portion of a sugar moiety. The sugar moiety may be or may contain one or more polysaccharides, such as complex branched dextran, such as dextran.

[0165] Solid supports may include one or more of the following: particles, resin, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. 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.

[0166] The kit may include one or more of the following: lysis or lysis / binding buffer, washing buffer, and elution buffer. The lysis or lysis buffer and / or washing buffer may contain a polar aprotic solvent of an aqueous nucleic acid purification buffer.

[0167] Lysis or lysis / binding buffers may contain high concentrations of a dissociative agent (e.g., guanidine salts or urea) and a surfactant, such as Triton X-100. Lysis or lysis / binding buffers may contain any 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.

[0168] The washing buffer may be or contain a purification buffer. 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, the washing buffer may represent an aqueous dilution of the purification buffer used for binding. The aqueous dilution may also contain other reagents that may not be present in the purification buffer used for binding, such as buffers, or buffers and inorganic salts. The washing buffer may contain a 1.1-fold to 5-fold dilution of the purification buffer; for example, the washing buffer may contain a 1.5-fold to 4-fold dilution of the purification buffer.

[0169] In some embodiments, the washing buffer may be the same as the purification buffer (or an aqueous dilution of the purification buffer). In other embodiments, the purification 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.

[0170] Elution buffers may contain water, Tris-HCl, EDTA, or combinations thereof. Conventional elution buffers can 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.

[0171] Another aspect provides a material composition. This material composition comprises (i) a solution containing the aqueous nucleic acid purification buffer of the present invention (e.g., according to the first aspect); and (ii) a nucleic acid solid support.

[0172] The solid support may contain surface hydroxyl functional groups. These surface hydroxyl functional groups may comprise a portion of a silanol, carboxyl, or sugar moiety. The surface hydroxyl functional groups may comprise a portion of a silanol moiety. The surface hydroxyl functional groups may comprise a portion of a carboxyl moiety. The surface hydroxyl functional groups may comprise a portion of a sugar moiety. The sugar moiety may be or may comprise one or more polysaccharides, such as complex branched dextran, like dextran.

[0173] Solid supports may include one or more of the following: particles, resin, beads, filters, cartridges, columns, arrays, membranes, chips, disks, or glass slides. 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.

[0174] Solution (i) may further contain lysis or lysis / binding buffer-related components, such as one or more of the following: a dissociation agent (e.g., guanidine salt or urea); a surfactant, such as Triton X-100; and one or more enzymes, such as lysozyme, proteolytic enzymes (e.g., proteinase K), etc. Solution (ii) may contain any components disclosed in connection with the methods, uses, and kits described herein.

[0175] The material composition may further include a sample, such as a pretreated or untreated biological sample, an environmental sample, or an enzyme reaction mixture, as described herein; the sample contains nucleic acids.

[0176] Nucleic acids can be DNA; such as plasmid DNA, cDNA, cfDNA, and / or genomic DNA. Nucleic acids can also be RNA; such as one or more of the following: mRNA (e.g., isolated from biological samples or...). in vitro Transcribed RNA, siRNA, microRNA, tRNA, or cfRNA.

[0177] Other aspects provide the use of the kits of the present invention in automated nucleic acid analysis platforms. The advantages of automated nucleic acid analysis systems are that they allow for the parallel analysis of large numbers of samples in a shorter time, eliminate the risk of manual handling errors during analysis, and require minimal operator intervention time. The kits of the present invention can be used in automated liquid handling, pipetting systems, or in both automated, multi-purpose, high-throughput integrated laboratory systems that include downstream processing capabilities. Exemplary automated nucleic acid analysis platforms used in the present invention may include, but are not limited to, liquid handling and automated systems such as the DreamPrep™ NAP workstation (TECAN), Fluent™ Automated Workstation (TECAN), Microlab Prep, NIMBUS, STAR, or VANTAGE pipetting platforms (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), or the COBAS™ 5800 system (Roche Diagnostics).

[0178] The seventh aspect provides a nucleic acid analysis device. This device includes an automated nucleic acid analysis platform and a reagent kit as described in the fourth aspect. The automated nucleic acid analysis platform includes a portion configured to contain a solid support.

[0179] Automated nucleic acid analysis platforms can be point-of-care testing (POC) instruments. POC instruments can be used for pathogen detection and / or biological warfare agent detection, and / or genetic disease detection. Pathogen detection or biological warfare agent detection may include viral or bacterial nucleic acid detection. An exemplary POC instrument includes ePlex. ® The system (obtained from GenMark Diagnostics, Inc., Carlsbad, California, USA) and Solana ® Instrument (obtained from Quidel, San Diego, California, USA). Further examples of point-of-care measuring instruments are provided in US 9,752,182 B2 and US 2016 / 0016171 A1, the contents of which are incorporated herein by reference.

[0180] Additional Implementation Plan

[0181] This invention and disclosure also include the subject matter of the following provisions: 1. An aqueous nucleic acid purification buffer, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.

[0182] 2. The purification buffer according to Clause 1, wherein a polar aprotic solvent is present in an amount of at least about 2% by weight.

[0183] 3. Purification buffer according to Clause 1 or Clause 2, wherein the polar aprotic solvent is present in an amount not exceeding about 80% by weight.

[0184] 4. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent is present in an amount between about 4% by weight and about 75% by weight.

[0185] 5. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent has a boiling point of more than 100°C at standard atmospheric pressure, and optionally more than 150°C at standard atmospheric pressure.

[0186] 6. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent contains 2, 3 or 4 heteroatoms selected from O and N; Optionally, the polar aprotic solvent contains 2 or 3 heteroatoms selected from O and N; and / or Optionally, the polar aprotic solvent consists of carbon atoms, hydrogen atoms, and 2, 3, or 4 heteroatoms selected from O and N.

[0187] 7. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent contains 4, 5, 6, 7, 8, 9 or 10 carbon atoms; Optionally, the polar aprotic solvent contains 5, 6, 7, or 8 carbon atoms; or Optionally, the polar aprotic solvent contains 6, 7, 8, 9 or 10 (e.g., 6, 7 or 8) carbon atoms.

[0188] 8. A purification buffer according to any of the preceding clauses, wherein the viscosity of the polar aprotic solvent at 20°C and standard atmospheric pressure is less than 50 cP; optionally, wherein the viscosity of the polar aprotic solvent at 20°C and standard atmospheric pressure is less than 25 cP.

[0189] 9. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent has a flash point of at least 50°C.

[0190] 10. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent is liquid at 0°C and standard atmospheric pressure.

[0191] 11. A purification buffer according to any of the preceding clauses, wherein the polar aprotic solvent is selected from one or more of the following: dihydro-L-glucoseenone, N-butylpyrrolidone-2-one, dipropylene glycol dimethyl ether, and N-formylmorpholine.

[0192] 12. A purification buffer according to any of the preceding clauses, wherein the purification buffer further comprises one or more of a dissociation agent, a surfactant, a buffer, an enzyme, an inorganic salt, and an antifoaming agent, or a combination thereof.

[0193] 13. A purification buffer according to any of the preceding clauses, wherein the purification buffer is a binding buffer and / or a washing buffer.

[0194] 14. A washing buffer according to Clause 13, wherein the washing buffer contains a 1.1 to 5 times dilution of the binding buffer.

[0195] 15. The purification buffer according to any one of Clauses 1 to 12 precipitates nucleic acids from solution onto a solid support, thereby providing a solid support for binding nucleic acids.

[0196] 16. As per the use of Clause 15, wherein the solid support comprises a surface hydroxyl functional group, optionally wherein the surface hydroxyl functional group comprises a portion of a silanol, carboxyl, or sugar moiety.

[0197] 17. As used pursuant to Clause 15 or 16, wherein the solid support comprises one or more of the following: particles, resin, box, beads, filter, column, array, membrane, chip, disk or slide.

[0198] 18. For any of the uses described in Clauses 15 to 17, wherein the solid support comprises beads, optionally monodisperse beads.

[0199] 19. As per the use of Clause 18, wherein the beads (optionally monodisperse) are magnetic.

[0200] 20. The use according to any of clauses 15 to 19 further includes washing the solid support bound to nucleic acid with a washing buffer.

[0201] 21. As per the purpose of Clause 20, the washing buffer is or contains a purification buffer.

[0202] 22. The use according to any of clauses 15 to 21 further includes contacting a solid support bound to nucleic acid with an elution buffer to separate the nucleic acid from the solid support.

[0203] 23. As per the use of Clause 22, the elution buffer comprises water, Tris-HCl, EDTA, or a combination thereof.

[0204] 24. As permitted by Clauses 22 to 23, wherein the isolated nucleic acid is subjected to 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, protein expression, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic preparations, and genome editing.

[0205] 25. As permitted by Clause 24, the amplification includes PCR, qPCR, reverse transcription, in vitro Transcription or isothermal amplification.

[0206] 26. As used in Clause 25, isothermal amplification includes loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple substitution amplification (MDA), recombinase polymerase amplification (RPA), single-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).

[0207] 27. For any of the uses in clauses 15 to 24, wherein the nucleic acid is DNA; optionally, one or more of the following: synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g., dsDNA or ssDNA), cDNA or cfDNA.

[0208] 28. For any of the uses of clauses 15 to 24, wherein the nucleic acid is RNA; optionally, one or more of the following: mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA or viral RNA (e.g., dsRNA or ssRNA).

[0209] 29. A method for processing nucleic acids, the method comprising: Samples containing nucleic acids were exposed to an aqueous medium containing a polar aprotic solvent in the presence of a solid support; and Nucleic acid is precipitated onto a solid support, thereby providing a solid support for binding nucleic acid.

[0210] 30. The aqueous medium containing a polar aprotic solvent, according to the method of Clause 29, is an aqueous nucleic acid purification buffer as defined in Clauses 1 to 14.

[0211] 31. The method according to Clause 29 or Clause 30, wherein the solid support comprises a surface hydroxyl functional group, optionally wherein the surface hydroxyl functional group comprises a portion of a silanol, carboxyl, or sugar moiety.

[0212] 32. The method according to any one of clauses 29 to 31, wherein the solid support comprises one or more of the following: particles, resin, box, beads, filter, column, array, membrane, chip, disk or slide; optionally, wherein the solid support comprises (optionally monodisperse) beads.

[0213] 33. The method according to any one of clauses 29 to 32, the method further comprising washing the solid support bound to nucleic acid with a washing buffer; optionally, wherein the washing buffer is or contains a purification buffer.

[0214] 34. The method according to any one of clauses 29 to 33, the method further comprising contacting a solid support for binding nucleic acid with an elution buffer to separate the nucleic acid from the solid support to provide isolated nucleic acid.

[0215] 35. The method according to any one of clauses 29 to 34, wherein the nucleic acid is subjected to one or more additional processes, optionally selected from detection, cloning, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, research, transfection, hybridization, cDNA synthesis, size separation, chromatography, mass spectrometry, pharmaceutical or therapeutic agents, and genome editing.

[0216] 36. The method according to any one of clauses 29 to 35, wherein the sample includes or is a pretreated or untreated biological sample, environmental sample or enzyme reaction mixture.

[0217] 37. The method according to Clause 36, wherein environmental samples include water samples (optionally, wastewater samples), soil samples, sediment samples, surface swabs, air-derived samples (such as air filter residues), cosmetics, food ingredients or food samples or combinations thereof.

[0218] 38. The method according to clause 36, wherein the enzyme reaction mixture comprises in vitro Transcription reaction mixture, reverse transcription reaction mixture, second-strand synthesis reaction mixture, amplification reaction mixture, library preparation reaction mixture, restriction reaction mixture, nucleic acid assembly reaction mixture, barcode reaction mixture.

[0219] 39. The method according to Clause 36, wherein the biological sample comprises one or more of the following: blood, bloodstains, umbilical cord blood, blood components (e.g., platelet concentrate), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, erythrocyte sedimentation rate (ESR) leukocytes, serum, plasma, saliva, saliva stains, oral cells, oral swabs, semen, semen stains, urine, feces, fecal stains, cigarette butts, chewing gum, formalin-fixed paraffin-embedded (FFPE) samples. 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 breath, 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 may optionally be in physiological buffers or transport media.

[0220] 40. The method according to any one of clauses 29 to 39, wherein the aqueous medium does not contain ethanol, isopropanol or 2-methyl-1,3-propanediol.

[0221] 41. The method according to any one of clauses 29 to 40, wherein the method further includes performing immediate detection.

[0222] 42. The method according to Clause 41, wherein point-of-care testing includes pathogen detection, biological warfare agent detection, or genetic disease detection.

[0223] 43. The method according to Clause 42, wherein pathogen detection or biological warfare agent detection includes viral or bacterial nucleic acid detection.

[0224] 44. A kit comprising an aqueous nucleic acid purification buffer as defined in any one of clauses 1 to 14, and a solid support.

[0225] 45. The kit according to Clause 44, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise a portion of a silanol, carboxyl, or sugar moiety; and / or

[0226] The solid support includes one or more of the following: particles, resin, beads, filters, boxes, columns, arrays, membranes or chips, disks or glass slides; optionally, the solid support includes (optionally monodisperse) beads.

[0227] 46. ​​The kit according to Clause 44 or Clause 45, further comprising one or more of the following: lysis buffer, washing buffer, and elution buffer; optionally, wherein the lysis buffer and / or washing buffer contains a polar aprotic solvent of an aqueous nucleic acid purification buffer.

[0228] 47. A composition comprising: (i) A solution comprising an aqueous nucleic acid purification buffer as defined in any one of clauses 1 to 14; and (ii) Nucleic acid solid support.

[0229] 48. The composition according to Clause 47, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise a portion of a silanol, carboxyl, or sugar moiety; and / or

[0230] The solid support includes one or more of the following: particles, resin, beads, filters, columns, boxes, arrays, membranes, chips, disks, or glass slides; optionally, the solid support includes (optionally monodisperse) beads.

[0231] 49. The composition according to Clause 47 or Clause 48, wherein the nucleic acid is DNA; optionally, plasmid DNA, cDNA, cfDNA and / or genomic DNA; or

[0232] The nucleic acid is RNA; optionally, one or more of the following: mRNA, siRNA, microRNA, tRNA, or cfRNA.

[0233] 50. Use of the kits described in any of Clauses 44 to 46 in an automated nucleic acid analysis platform.

[0234] 51. A nucleic acid analysis device, the nucleic acid analysis device comprising: Automated nucleic acid analysis platform; and According to any of clauses 44 to 46, the reagent kit The automated nucleic acid analysis platform includes a section that is configured to accommodate a solid support.

[0235] 52. A nucleic acid analysis device according to Clause 51, wherein the automated nucleic acid analysis platform is a point-of-care testing instrument.

[0236] 53. A nucleic acid analysis device pursuant to Clause 52, wherein the point-of-care testing instrument is suitable for pathogen detection and / or biological warfare agent detection, and / or genetic disease detection.

[0237] Example

[0238] Example 1: A scheme for preparing viral nucleic acid (vNA) using silane beads

[0239] Reagents: Dynabeads™ MyOne SILANE silane magnetic beads (Thermo Fisher Scientific).

[0240] Aqueous NA purification buffer containing guanidine salts and Tween.

[0241] Trigger: A nonprotic solvent for the nucleic acid purification buffer, as shown in Table 1 of the implementation plan. Isopropanol (IPA) is used as an alternative trigger for the positive control.

[0242] 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.

[0243] Washing 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.

[0244] Elution buffer: Tris pH 8.

[0245] LB buffer contains guanidine salts, ionic or nonionic detergents (e.g., Tween, such as Tween20), pH 5-9.

[0246] Proteinase K, 20 mg / mL.

[0247] Table 1: Trigger parameters for trigger testing: Add 150 µL of trigger at the following dilution in step 6 of the protocol.

[0248] Consumables and equipment: Magnetic Separator: DynaMag™-2 Magnet, SKU 12321D (Thermo Fisher Scientific). This device provides a holder for microtubes with a built-in magnet at the bottom for effective separation of magnetic beads from the supernatant.

[0249] 1.5 mL microtube.

[0250] Test tube roller / rotator.

[0251] Microtube heating block (preheated to the required pyrolysis and / or elution temperature).

[0252] Microtubule benchtop centrifuge.

[0253] 1000 µL, 200 µL and 100 µL pipettes.

[0254] Timer.

[0255] Hot mixer / oscillator.

[0256] Optional equipment for efficient manual processing: o Laboratory vacuum pump used to remove supernatant; o Disposable glass Pasteur pipette with long capillary tip.

[0257] process: 1. Allow the buffer solution to reach room temperature before use. Remove from the refrigerator. Silane magnetic beads Vortex the bottle for 20 seconds to resuspend the beads. Before use in step 6, rotate the bottle on a roller / rotator for 20 minutes at room temperature.

[0258] 2. Add 200 µL of serum / plasma / UTM (universal transport medium) sample to a 1.5 mL test tube.

[0259] 3. Add 50 µL Proteinase K Use short-duration pulsed vortex mixing.

[0260] 4. Add 300 µL LB buffer Mix using a brief pulsed vortex and then incubate at room temperature for 10 minutes. For a hot mixer / oscillator, mix at 900 rpm for 10 minutes.

[0261] 5. Add 25 µL Dynabeads MyOne SILANE (1 mg) or Dynabeads MyOne Carboxylic Acid (1 mg) mg). Use brief pulsed vortex mixing.

[0262] 6. Add 150 µL Trigger Gently add to the lysis buffer. Mix using a brief pulsed vortex. Incubate the tube on a vortex mixer / roller for 10 minutes at room temperature. For a hot mixer / shaker, mix at 900 rpm for 10 minutes.

[0263] 7. Place the test 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.

[0264] 8. Carefully remove using a 1000 µL pipette or vacuum pump. pyrolysis products Discard it. Remove the test tube from the magnet.

[0265] 9. Cleaning buffer 1. First cleaning: Add 800 µl Washing buffer 1 Vortex until the beads are completely resuspended. Place the test tube on a DynaMag™-2 magnet. Allow the beads to adhere to the magnet for 1 to 2 minutes.

[0266] 10. Carefully remove using a 1000 µL pipette or vacuum pump. Washing buffer 1 And discard it.

[0267] 11. Cleaning buffer 1. Second cleaning: Perform a second cleaning by repeating steps 9 to 10.

[0268] Note: Use the same volume as in step 9. Washing buffer 1 .

[0269] 12. Cleaning buffer 2. First cleaning: Remove the test tube from the magnet and add 500 µL Cleaning buffer Liquid 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.

[0270] 13. Allow the beads to adhere to the magnet for 1 minute. Carefully remove them from the 1.5 mL test tube using a pipette with a 1000 µL tip or a vacuum pump. Washing buffer 2 And discard it.

[0271] 14. Cleaning buffer 2. Second wash: Add 500 µL Washing buffer 2 .

[0272] 15. Remove the test tube from the magnet. Mix by vortexing / shaking until the beads are completely resuspended, and then collect the bead solution by brief centrifugation (1-second pulse). Be careful not to let the beads clump together.

[0273] 16. Place the test tube back onto the DynaMag™-2 magnet. Allow the beads to adhere to the magnet for 30 seconds, or until they are completely removed. Carefully remove and discard the supernatant using a pipette with a 1000 µL tip or a vacuum pump.

[0274] 17. To remove any stagnant liquid between the beads, tap the holder / magnet containing the test tube on the top of the workbench with moderate force 3 to 5 times. Use a small pipette to aspirate and discard the last drop. All liquid should be removed before elution.

[0275] 18. Add 100 µL Elution buffer .

[0276] 19. Vortex for 20 seconds to 1 minute until the beads are completely resuspended. Collect the bead solution using a brief centrifugation (1-second pulse). Be careful not to allow the beads to clump together.

[0277] 20. Incubate in a preheated block at 80°C for 5 minutes. For a hot mixer / oscillator, mix at 80°C and 1500 rpm for 5 minutes.

[0278] 21. Vortex for 15 seconds, then collect the bead solution by brief centrifugation (1-second pulse).

[0279] 22. Place the test tube on the DynaMag™-2 magnet. Allow the beads to adhere to the magnet for 15 seconds.

[0280] 23. Transfer the eluted nucleic acids to a new 1.5 mL test tube. Store at an appropriate temperature (e.g., DNA: 4°C to -80°C).

[0281] Example 2: A scheme for preparing viral nucleic acid (vNA) using carboxylic acid beads

[0282] Example 2 follows the same scheme as Example 1, but replaces the "silane magnetic beads" with the following reagents: Carboxylic acid magnetic beads: Dynabeads™ MyOne™ Carboxylic Acid (Thermo Fisher Scientific).

[0283] Example 3: Evaluation of purification buffers containing polar aprotic solvents using silane and carboxylic acid beads

[0284] 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, 1.4 e5 pfu and 1.5 e6 copies of M13 phage (1.4 e5 pfu) and MS2 RNA (1.5 e6 copies) were infused into 200 µL of human plasma, respectively, before sample processing, and then processed according to the protocols of Examples 1 and 2. The eluted samples were then analyzed by qPCR to quantify the recovery rate of M13 DNA, or by RT-qPCR to quantify the recovery rate of MS2 RNA.

[0285] Figure 3 provides the results demonstrating the recovery of nucleic acids from silanol-functionalized stationary phases using a nonprotic solvent selected from Cyrene™, Proglyde™, TamiSolve™, or N-formylmorpholine (N-FM) in binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. (A) shows the MS2 RNA recovery rate on silane beads, while (B) shows the M13 DNA recovery rate on silane beads.

[0286] Figure 4 provides the results demonstrating the efficiency of binding and recovering nucleic acids from carboxylic acid functionalized immobilization using a nonprotic solvent selected from Cyrene™, Proglyde™, TamiSolve™, or N-formylmorpholine (N-FM) in binding buffer, with the protic solvent isopropanol (IPA) used as a positive control. (A) shows the recovery rate of MS2 RNA on carboxylic acid beads, while (B) shows the recovery rate of M13 DNA on carboxylic acid beads.

[0287] As shown in Figures 3 and 4, all purification buffers containing polar aprotic solvents resulted in NA precipitation / binding to silane or carboxylic acid beads, but to varying degrees. Data indicate that Proglyde™ and TamiSolve™ show promise as aprotic solvents for nucleic acid binding buffers or washing buffers for both silane and carboxylic acid surfaces, but were more effective on carboxylic acid surfaces in the tested buffer compositions. Cyrene™ was most effective on silane beads (M13 data for Cyrene™ on silane beads are not shown). N-formylmorpholine exhibited good activity towards DNA targets on silane and lower efficiency towards RNA targets on silane, suggesting its potential for selective DNA purification in samples containing both DNA and RNA.

[0288] Example 4: Workflow based on a completely aprotic solvent

[0289] The efficiency of an exemplary purification workflow was evaluated, in which all buffers contained polar aprotic solvents for nucleic acid purification.

[0290] To investigate the efficiency of these buffers in triggering the binding of nucleic acids to carboxylic acid-functionalized surfaces and in washing away bound nucleic acid molecules, approximately 100 copies of a 120 bp DNA model and MS2 RNA were incorporated into 200 μL of human plasma, and then processed according to the protocols of Examples 1 and 2.

[0291] Replace isopropanol and / or ethanol with TamiSolve™ and / or 2-butoxyethanol as shown in Table 2 below. Analyze the resulting eluent by qPCR or RT-qPCR.

[0292] Table 2: Solvents and buffers used in the complete aprotic workflow and other extractions

[0293] Figures 5a and 5b show the PCR results. Figures 6A and 6B provide the recovery percentage calculated based on the copy number incorporated into the serum.

[0294] As shown in Figures 5B and 6B, for DNA targets, the fully alternative TamiSolve™ workflow produced equally sensitive DNA target separation as the standard alcohol workflow (extraction number 7 in the 120 bp DNA model diagram).

[0295] For RNA targets, a slightly less optimal separation was achieved using a workflow that completely replaced TamiSolve™ (extraction number 7 in the MS2 RNA diagram, Figure 5A). However, better RNA recovery was obtained by using TamiSolve™ to trigger binding in wash buffer 1 and 2-butoxyethanol in wash buffer 2 (extraction number 6). Figure 5 A and Figure 6B).

Claims

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

2. The purification buffer of claim 1, 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.

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

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

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

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

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

8. The purification buffer of any preceding claim, wherein the polar aprotic solvent has a flash point of at least 50°C; and / or wherein the polar aprotic solvent is a liquid at 0°C and standard atmospheric pressure.

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

10. The purification buffer of any preceding claim, wherein the purification buffer further comprises one or more or a combination of a chaotropic agent, a surfactant, a buffer, an enzyme, an inorganic salt, an antifoam agent.

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

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

13. Use of the purification buffer of any one of claims 1 to 12 to precipitate a nucleic acid from a solution onto a solid support, thereby providing a solid support bound nucleic acid.

14. The use of claim 13, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise silanols, carboxyl groups, or part of a sugar moiety.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

30. A composition of matter comprising: (i) a solution comprising an aqueous nucleic acid purification buffer as defined in any one of claims 1 to 12; and (ii) a nucleic acid solid support.

31. The composition of claim 30, wherein the solid support comprises surface hydroxyl functional groups, optionally wherein the surface hydroxyl functional groups comprise a portion of a silanol, a carboxyl, or a sugar moiety; and / or wherein the solid support comprises one or more of a particle, a resin, a bead, a filter, a column, a cartridge, an array, a membrane, or a chip; optionally wherein the solid support comprises a (optionally monodisperse) bead.

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

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

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

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