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WO2025078670A1 - Isolement successif d'acides nucléiques - Google Patents

Isolement successif d'acides nucléiques Download PDF

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Publication number
WO2025078670A1
WO2025078670A1 PCT/EP2024/078805 EP2024078805W WO2025078670A1 WO 2025078670 A1 WO2025078670 A1 WO 2025078670A1 EP 2024078805 W EP2024078805 W EP 2024078805W WO 2025078670 A1 WO2025078670 A1 WO 2025078670A1
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Prior art keywords
sample
buffer
solid support
nucleic acid
dna
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English (en)
Inventor
Hannah KIM LINDSTRÖM
Frauke HENJES
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Life Technologies AS
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Life Technologies AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • AHPrep® DNA/RNA/Protein a lysate is passed through an AHPrep DNA spin column, which, in combination with a high-salt buffer, allows selective binding of genomic DNA. The column is then washed, and the bound DNA is eluted. Ethanol is then added to the flow-through from the spin column to provide appropriate binding conditions for RNA. The sample is applied to an RNeasy spin column where total RNA binds to the membrane.
  • Qiagen’s AHPrep® DNA/RNA/Protein Kit therefore requires both the lysis binding buffer chemistry and the solid phase to be changed in order to facilitate sequential DNA and RNA extraction.
  • a first aspect of the invention provides a method of sequentially isolating DNA and RNA from a sample, the method comprising steps a) to d): a) contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA- bound solid support; b) removing the DNA-bound solid support from the sample; c) contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and d) removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.
  • the polar aprotic solvent may be present in an amount of at least about 2 %wt.
  • the polar aprotic solvent may be present in an amount of not more than about 80 %wt.
  • the polar aprotic solvent may be present in an amount of from between about 4 %wt to about 75 %wt.
  • the polar aprotic solvent may comprise 2, 3 or 4 heteroatoms selected from O and N.
  • the polar aprotic solvent may comprise 2 or 3 heteroatoms selected from O and N.
  • the polar aprotic solvent may comprise 2 or 3 O atoms.
  • the polar aprotic solvent may comprise 1 or 2 O atoms and 1 or 2 N atoms.
  • the polar aprotic solvent may comprise 2 O atoms.
  • the polar aprotic solvent may comprise 3 O atoms.
  • the polar aprotic solvent may comprise 1 O atom and 1 or 2 N atoms.
  • the polar aprotic solvent may comprise 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • the polar aprotic solvent may comprise 5, 6, 7, or 8 carbon atoms, e.g. the polar aprotic solvent may comprise 6 or 8 carbon atoms.
  • the polar aprotic solvent may comprise 5 carbon atoms.
  • the polar aprotic solvent may comprise 6 carbon atoms.
  • the polar aprotic solvent may comprise 7 carbon atoms.
  • the polar aprotic solvent may comprise 8 carbon atoms.
  • the polar aprotic solvent may have a viscosity of less than 50 cP at 20 °C and 1 atm.
  • the polar aprotic solvent may have a viscosity of less than 40 cP or 30 cP at 20 °C and 1 atm, e.g. the polar aprotic solvent may have a viscosity of less than 25 cP at 20 °C and 1 atm.
  • the polar aprotic solvent may have a flashpoint of at least 50 °C.
  • the polar aprotic solvent may be liquid at 0 °C and 1 atm.
  • the polar aprotic solvent may be non-flammable, non-viscous, EHS-friendly, and/or biorenewable.
  • the polar aprotic solvent may be an environmental, health and safety (EHS) friendly solvent.
  • EHS environmental, health and safety
  • the polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N- Formylmorpholine.
  • the polar aprotic solvent may be selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, and dipropylene glycol dimethyl ether.
  • the polar aprotic solvent is N-butylpyrrolidin-2-one.
  • the purification buffer may further comprise one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof.
  • the purification buffer may comprise two or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent, e.g. the purification buffer may comprise three or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent.
  • the purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of an alcohol or a polyol. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of alcohol or polyol may be preferred.
  • the purification buffer may comprise a modest amount (e.g. less than about 20%, less than about 10%, or less than about 5%) of a polyethylene glycol PEG, a TEG, or an LPA. Purification buffer with no more than a modest amount (e.g. with less than about 5%, or with less than about 2%) of a PEG, a TEG, or an LPA may be preferred.
  • the wash buffer may represent an aqueous dilution of the binding buffer.
  • the aqueous dilution may also comprise other reagents that may not be present in the binding buffer, such as: a buffering agent, or a buffering agent and an inorganic salt.
  • the wash buffer may comprise a 1.1 to 5 times aqueous dilution of the binding buffer, e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the binding buffer.
  • a workflow or kit could comprise both a binding buffer of the present invention and a wash buffer of the present invention, where the binding buffer and wash buffers have different compositions, i.e. the wash buffer is neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.
  • the purification buffer does not contain ethanol, isopropanol or 2- methyl-1,3- propanediol. In embodiments, the purification buffer does not contain dimethylsulfoxide (DMSO). In embodiments, the purification buffer does not contain ethanol, isopropanol, 2-methyl-1,3- propanediol, or DMSO. In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol). In embodiments, the purification buffer does not contain an alcohol (including any diol or polyol) or DMSO. In embodiments, the purification buffer does not contain a salt or a chaotrope.
  • DMSO dimethylsulfoxide
  • the method may initially comprise suspending or dissolving the solid sample in a suitable buffer, e.g. the aqueous nucleic acid purification buffer.
  • the method may be a method of sequentially isolating protein, DNA and RNA from the sample, the method further comprising isolating protein before performing step a).
  • Isolating protein from the sample may comprise a targeted affinity protein separation, such as immunoprecipitation.
  • An affinity protein separation is conveniently carried out by any appropriate method using a solid phase with appropriate surface properties. Appropriate methods will depend on the type of target proteins.
  • specific proteins may be isolated using a solid support, which has an appropriate binding partner/ ligand attached to its surface. This thus applies well known principles of affinity separation of proteins, as widely described in the prior art. Any such standard and well known methods may be used or adapted for the present invention.
  • Affinity reagents are well known to the skilled person, may be available commercially or produced individually, e.g. by using an antibody coupling kit which enables the user to covalently couple antibodies or antigens to a solid support, such as DynabeadsTM magnetic beads, for the use in immunoprecipitation workflows.
  • a solid phase with more general surface binding properties can be selected, e.g. a solid phase which has surface chemistry which effects classical chromatographic interactions such as ion exchange (including both anion exchange and cation exchange), reverse phase interactions, or hydrophobic interactions.
  • Such surfaces may be conveniently provided on magnetic beads.
  • the use of these more general surfaces conveniently allows the fractionation of proteins in the sample into subsets depending on the structure and properties (charge, hydrophobicity, etc.) of the proteins present.
  • Some of these generic protein enrichment methods use weak to strong anionic or cationic surface chemistry.
  • Such general solid phase surfaces can be prepared using conventional techniques which are standard and well documented in the art of column chromatography.
  • isolating protein from the sample may, e.g., comprise contacting the sample with a solid support comprising ionizable surface groups, lowering the pH of the sample to ionize the surface groups thereby causing the ionized surface groups to selectively bind both DNA and RNA to form a nucleic acid-bound solid support.
  • the nucleic acid-bound solid support may then be removed from the protein, optionally washed (e.g. with a wash buffer) and contacted with an elution buffer, thereby separating the DNA and RNA from the solid support.
  • SAX strong anionic exchange
  • SAX strong anionic exchange
  • a high salt e.g. 1M NaCI
  • the solution containing the DNA and RNA that did not bind to the solid support will form the (protein free) sample used in step a).
  • This sample will typically be added to the aqueous nucleic acid purification buffer or vice versa.
  • plant or fungal cells or solid animal tissues may require more vigorous treatment such as, for example, grinding in liquid nitrogen, heating in the presence of detergent, alkaline lysis in the presence of detergent or freeze/thawing cycles.
  • procedures for freeing nucleic acid and protein are chosen such that the particular nucleic acid and protein species which are to be isolated in the methods of the invention remain sufficiently intact, e.g. are not substantially degraded and the lysis condition does not negatively affect any antibody or antigen or their affinity for each other if an affinity method is used in the optional protein isolation step.
  • any lysis step is performed before step a) or during step a). It may be that the lysis step is performed before isolating protein from the sample. It may be that the lysis step is performed after isolating protein from the sample. A lysis step may also be performed between the targeted isolation of two or more different proteins or between a targeted and a more general protein isolation.
  • the lysis step may comprise adding a lysis buffer to the sample. After combining sample and lysis buffer additional lysis steps may be performed, like agitation, heating and/or incubation. It may be that the lysis buffer is the aqueous nucleic acid purification buffer.
  • the binding step a) by adding the polar aprotic solvent and the solid support comprising surface silanol groups to the lysis buffer and provide a one-step lysis and binding workflow.
  • the polar aprotic solvent and the solid support comprising surface silanol groups may be added prior to combining sample and lysis buffer or after combining sample and lysis buffer and optional additional lysis steps like agitation and/or incubation.
  • the lysis or lysis/nucleic acid purification buffer may comprise a high concentration of chaotrope (for example a guanidinium salt or urea) and surfactant, e.g. Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, Octyl-beta- Glucoside, Octylthio Glucoside, SDS (sodium dodecyl sulfate), CHAPS, and/or CHAPSO, preferably Tween-20.
  • the lysis or lysis/binding buffer may comprise any of the components disclosed in connection with methods, uses and compositions herein.
  • the lysis buffer may comprise enzymes, e.g. lysozyme, proteolytic enzymes (such as Proteinase K), and the like.
  • the method may further comprise contacting the sample with a proteolytic enzyme, e.g. Proteinase K, prior to step a), provided that said proteolytic enzyme/Proteinase K is not added until after the (optional) protein isolation step. It may be that the method further comprises contacting the sample with Proteinase K and a lysis buffer prior to step a), provided that said Proteinase K and lysis buffer are not added until after the (optional) protein separation.
  • a proteolytic enzyme e.g. Proteinase K
  • Steps a) and b) are carried out before steps c) and d). It may be however that steps c) and d) are carried out before steps a) and b).
  • the method will comprise adding the solid support comprising surface carboxyl groups to the sample after step b).
  • Step b) may further comprise optional step b2): washing the DNA-bound solid support with a wash buffer.
  • Step b2) may comprise washing the DNA-bound solid support at least once.
  • Step b2) may comprise washing the DNA-bound solid support at least twice, e.g. at least three times, each time with fresh wash buffer.
  • the wash buffer comprises the purification buffer.
  • the wash buffer may be the same as (or be an aqueous dilution of) the purification buffer.
  • the purification buffer and wash buffers have different compositions, i.e. the wash buffer is neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.
  • Binding conditions, which precipitate the nucleic acid onto the solid support may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support.
  • the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding.
  • the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer, e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.
  • Step b2) may comprise washing the DNA-bound solid support with wash buffer 1.
  • Wash buffer 1 may comprises a nucleic acid purification buffer. It may be that wash buffer 1 comprises at least one polar aprotic solvent (e.g., the polar aprotic solvent that is present in the aqueous nucleic acid purification buffer, such as dihydrolevoglucosenone, N- butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N-Formylmorpholine; e.g. N- butylpyrrolidin-2-one).
  • polar aprotic solvent e.g., the polar aprotic solvent that is present in the aqueous nucleic acid purification buffer, such as dihydrolevoglucosenone, N- butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, or N-Formylmorpholine; e.g. N- butylpyrrolidin-2-one).
  • Wash buffer 1 may comprise from about 25 to about 75% by volume the organic solvent (e.g., isopropyl alcohol). Wash buffer 1 may comprise from about 35 to about 65% by volume the organic solvent (e.g., isopropyl alcohol). Wash buffer 1 may comprise from about 45 to about 55% by volume the organic solvent (e.g., isopropyl alcohol).
  • the organic solvent may be an alcohol, e.g. a Ci-Ce alcohol, diol (e.g. methylpropyl diol), polyol, or oligoethylene glycol. It may be that the organic solvent is a Ci-Ce alcohol.
  • the alcohol may be ethanol, or isopropanol.
  • Wash buffer 2 may comprise from about 50 to about 90% by volume of an alcohol (e.g., ethanol). Wash buffer 2 may comprise from about 60 to about 80% by volume of an alcohol (e.g., ethanol). It may be that the organic solvent is a glycol ether, e.g. a C2-C10 glycol ether.
  • the glycol ether may be 2-butoxyethanol.
  • wash buffer 2 comprises less than about 50% by volume N- butylpyrrolidin-2-one. It may be that wash buffer 2 comprises less than about 25% by volume N-butylpyrrolidin-2-one. It may be that wash buffer 2 comprises less than about 5% by volume N-butylpyrrolidin-2-one. It may be that wash buffer 2 does not comprise N- butylpyrrolidin-2-one.
  • Step b) may further comprise optional step b3): contacting the DNA-bound solid support with an elution buffer, thereby separating the DNA from its solid support.
  • the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.
  • a conventional elution buffer may be used, for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 Mm Tris-HCI buffer at pH 8.0.
  • Step d) may further comprise optional step d2): washing the RNA-bound solid support with a wash buffer.
  • Step d2) may comprise washing the RNA-bound solid support at least once.
  • Step d2) may comprise washing the RNA-bound solid support at least twice, e.g. at least three times, each time with fresh wash buffer.
  • the wash buffer is or comprises the purification buffer.
  • the wash buffer may be the same as (or be an aqueous dilution of) the purification buffer.
  • the purification buffer and wash buffers have different compositions, i.e. the wash buffer is neither the same as the binding buffer, nor does it represent an aqueous dilution of the binding buffer.
  • Binding conditions, which precipitate the nucleic acid onto the solid support may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support.
  • the wash buffer may represent an aqueous dilution of the purification buffer that is used for binding.
  • the wash buffer may comprise a 1.1 to 5 times dilution of the purification buffer, e.g. the wash buffer may comprise a 1.5 to 4 times dilution of the purification buffer.
  • Step d) may further comprise optional step d3): contacting the RNA-bound solid support with an elution buffer, thereby separating the RNA from its solid support.
  • the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.
  • a conventional elution buffer may be used, such as for example an aqueous Tris buffer at pH greater than 7, e.g. a 10 mM Tris-HCI buffer at pH 8.0.
  • the method comprises contacting the sample with a protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample.
  • the method may comprise contacting the sample with the immobilized protein affinity reagent in the aqueous nucleic acid purification buffer that is used for the subsequent sequential isolation of DNA and RNA.
  • the method may further comprise washing the protein-bound solid support with a wash buffer.
  • the method may comprise washing the protein-bound solid support at least once.
  • the method may comprise washing the proteinbound solid support at least twice, e.g. at least three times, each time with fresh wash buffer.
  • the wash buffer is or comprises a physiological buffer, PBS, HEPES, Tris, NH4OAC, etc with or without salts (e.g. KOAc, NaCI, MgCh, KOI), with or without ionic or non-ionic, anionic or cationic, or zwitterionic detergents, with or without reducing agents, with or without EDTA/EGTA etc., depending on the nature of the affinity reagent and the target protein as well as subsequent uses of the isolated target protein.
  • salts e.g. KOAc, NaCI, MgCh, KOI
  • the method may further comprise contacting the protein-bound solid support with an elution buffer, thereby separating the protein from its solid support.
  • the elution buffer comprises an aqueous salt solution, such as saline, or solution comprising glycine-HCI at a pH of around 2.5-3.0.
  • the separated protein, DNA and/or RNA may be further subjected to one or more additional processes like identification and/or quantification.
  • additional downstream processes may be selected from but not limited to e.g. microarray analysis, detection, cloning, restriction, nucleic acid synthesis and/or assembly, epigenetic analysis, sequencing, amplification, transfection, hybridisation, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.
  • the amplification may comprise PGR, qPCR, digital PGR (dPCR), reverse transcription, in vitro transcription, or isothermal amplification.
  • the isothermal amplification may comprise loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA) (for an overview see “Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", Infection and Drug Resistance 2020:13, 455-483 and references therein).
  • LAMP loop-mediated isothermal amplification
  • RCA rolling circle amplification
  • HDA heli
  • the sequencing may comprise next generation sequencing.
  • “Next-generation sequencing” and “high-throughput sequencing” are sequencing techniques that parallelize the sequencing process, producing a high number of sequences at once.
  • the nextgeneration sequencing methods may comprise single molecule real-time sequencing (e.g., Pacific Biosciences), ion semiconductor sequencing (e.g., Ion Torrent), pyrosequencing (e.g., 454 Life Sciences), sequencing by ligation (e.g., SOLiD sequencing of Applied Biosystems, Thermo Fisher Scientific), sequencing by synthesis and reversible terminator (e.g., Illumina), reversible dye terminator-based sequencing (e.g.
  • the separated RNA is further subjected to one or more additional processes selected from but not limited to e.g. reverse transcription, microarray analysis, qPCR, dPCR and next-generation sequencing.
  • the method may further comprise performing point-of-care (POC) detection.
  • the point-of-care detection may comprise e.g. testing for clinical markers or drugs, pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection.
  • the pathogen detection or biological warfare agent detection may comprise viral, bacterial, single-celled fungi or protozoan nucleic acid and/or protein detection.
  • the POC detection may be a single or a multiplex detection.
  • the testing for clinical markers or drugs as well as the genetic disease detection may include prenatal testing.
  • Any isolated protein(s) may be further subjected to one or more additional processes. After a targeted affinity separation, the protein may be quantified. After a non targeted separation proteins may be further separated, identified and/or quantified using methods well known in the art.
  • the one or more additional processes may be selected from e.g. protein microarray analysis, mass spectrometry, light scattering, immunoassays like e.g. ELISA or Western blot, protein sequencing, enzymatic activity and/or other methods known in the art.
  • the solid support comprising surface silanol groups may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide.
  • the solid support comprising surface silanol groups may comprise beads, for example monodisperse beads.
  • the (optionally monodisperse) beads may be magnetic.
  • the solid support comprising surface silanol groups may be a component of a robotic liquid handling platform.
  • the solid support comprising surface carboxyl groups may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide.
  • the solid support comprising surface carboxyl groups may comprise beads, for example monodisperse beads.
  • the (optionally monodisperse) beads may be magnetic.
  • the solid support comprising surface carboxyl groups may be a component of a robotic liquid handling platform.
  • the solid support to which the protein affinity reagent e.g. antigen, ligand, antibody, peptide, aptamer, lectin, carbohydrate, metal ions, engineered protein scaffold, a substrate and/or an inhibitor or metabolite of the target protein is immobilized may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide.
  • the solid support to which the protein affinity reagent is immobilized may comprise beads, for example monodisperse beads.
  • the (optionally monodisperse) beads may be magnetic.
  • the solid support to which the protein affinity reagent is immobilized may be a component of a robotic liquid handling platform.
  • the solid support comprising ionizable surface groups may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide.
  • the solid support comprising ionizable surface groups may comprise beads, for example monodisperse beads.
  • the (optionally monodisperse) beads may be magnetic.
  • the solid support comprising ionizable surface groups may be a component of a robotic liquid handling platform.
  • the RNA may, for example, be one or more of mRNA (e.g. isolated from a biological sample or in v/tro-transcribed), siRNA, microRNA, tRNA, cfRNA, rRNA, viral RNA (e.g. dsRNA or ssRNA), snRNA, or ctRNA.
  • mRNA e.g. isolated from a biological sample or in v/tro-transcribed
  • siRNA e.g. isolated from a biological sample or in v/tro-transcribed
  • microRNA e.g. isolated from a biological sample or in v/tro-transcribed
  • tRNA e.g. isolated from a biological sample or in v/tro-transcribed
  • rRNA e.g. dsRNA or ssRNA
  • viral RNA e.g. dsRNA or ssRNA
  • snRNA e.g. snRNA
  • ctRNA e
  • the sample may comprise or be a pre-treated or untreated biological sample, clinical or environmental sample, or an enzymatic reaction mixture.
  • the sample may be or comprise a pre-treated or untreated biological sample.
  • the biological sample may be in a physiological buffer or transport medium.
  • the biological sample may be a harvested or biopsied sample or a cultured sample.
  • the sample may be or comprise an environmental sample.
  • the sample may be or comprise an enzymatic reaction mixture.
  • the biological sample may be any suitable biological sample.
  • Exemplary biological samples may comprise but are not limited to one or more of blood, blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin- fixed paraffin-embedded (FFPE) sample, biopsy (e.g.
  • FFPE formalin- fixed paraffin-embedded
  • the biological sample may comprise exosomes.
  • the biological sample may be a fluid biological sample. In some instances, the biological sample may be a clinical sample. In some instances, the sample may
  • the environmental sample may comprise a water sample (such as a wastewater sample, swimming pool water sample, ocean water sample), a soil sample, a sediment sample, a surface swab, an air derived sample (such as an air filter residue), a cosmetic, a food ingredient or a food sample, or a combination thereof.
  • a water sample such as a wastewater sample, swimming pool water sample, ocean water sample
  • a soil sample such as a soil sample, a sediment sample, a surface swab
  • an air derived sample such as an air filter residue
  • cosmetic such as a food ingredient or a food sample, or a combination thereof.
  • the enzymatic reaction mixture could be any such mixture that may contain nucleic acid sequences.
  • the enzymatic reaction mixture may comprise in vitro transcription reaction mixture, a reverse transcription reaction mixture, a second strand synthesis reaction mixture, a nucleic acid assembly reaction mixture, an amplification reaction mixture, a library preparation reaction mixture, a restriction reaction mixture, a nucleic acid assembly reaction mixture, or a barcoding reaction mixture.
  • the method of the present invention may advantageously allow for the sequentially extraction of RNA and DNA present in a sample at very low numbers in a large sample volume (e.g. cfDNA or cfRNA, ctDNA or ctRNA in plasma) or from small samples (e.g. single- or few-call samples, as is often the case with CTCs). It may therefore that the sample comprises DNA and/or RNA at levels ranging from about 1 picogram nucleic acid/mL (or even lower) to about 1 microgram of nucleic acid/mL (or even higher).
  • qPCR or RTqPCR may be used for detection and quantification of DNA/RNA in the below picogram range. In the pico to nanogram range fluorescent nucleic acid intercalating dyes may be used for detection and quantification.
  • the specific DNA and RNA to be isolated will vary based on the sample.
  • the sample may comprise CTCs
  • the DNA may comprise gDNA and the RNA may comprise total RNA. It may be that the sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.
  • a second aspect of the invention provides a kit comprising: a first solid support comprising surface silanol groups; a second solid support comprising surface carboxyl groups; and an aqueous nucleic acid purification buffer comprising a polar aprotic solvent.
  • the kit may additionally comprise components for one or more targeted protein isolations and/or non-targeted protein isolations, such as a third solid support comprising an immobilized protein affinity reagent, such as e.g. a tosylactivated surface to bind an antibody directed at a target protein to be isolated or a solid support having a surface that is suitable to bind to a target protein, subgroup of proteins or all proteins in a sample.
  • a third solid support comprising an immobilized protein affinity reagent, such as e.g. a tosylactivated surface to bind an antibody directed at a target protein to be isolated or a solid support having a surface that is suitable to bind to a target protein, subgroup of proteins or all proteins in a sample.
  • an immobilized protein affinity reagent such as e.g. a tosylactivated surface to bind an antibody directed at a target protein to be isolated or a solid support having a surface that is suitable to bind to a target protein, subgroup
  • the aqueous nucleic acid purification buffer and a polar aprotic solvent may be as defined in the first aspect above.
  • a fourth aspect of the invention provides a nucleic acid analysis apparatus, comprising: an automated nucleic acid analysis platform; and a kit of any of the second aspect of the invention, wherein the automated nucleic acid analysis platform comprises a portion configured to house the solid support.
  • the automated nucleic acid analysis platform may be a point-of care assay instrument.
  • the point-of-care assay instrument may be adapted for pathogen detection, and/or biological warfare agent detection, clinical markers or drugs and/or genetic disease detection.
  • the pathogen detection or biological warfare agent detection may comprise viral, bacterial, single-celled fungi or protozoan nucleic acid detection.
  • the point-of-care assay instrument is adapted for cancer detection.
  • the testing for clinical markers, pathogens or drugs as well as the genetic disease detection may include prenatal testing.
  • the present invention has multiple applications in laboratory research, human and veterinary medicine, public health and sanitation, forensics, anthropological studies, environmental monitoring, and industry.
  • Such applications include, without limitation, bacterial and viral detection and typing, microbial drug resistance screening, viral load assays, genotyping, infection control and pathogen screening (of, e.g., blood, tissue, food, cosmetics, water, soil, and air), pharmacogenomics, detection of cell-free DNA in plasma, white cell counting, and other fields where preparation and analysis of DNA from biological samples is of interest.
  • Figure 1 provides an exemplary schematic illustrating a general process for the sequential isolation of protein, DNA and RNA from a sample.
  • Figure 2 provides an exemplary schematic illustrating a more detailed process for the sequential isolation of protein, DNA and RNA from a liquid biopsy sample.
  • Figure 3 is a bar chart showing the recovery of nucleic acids from silane and carboxylic acid functionalised stationary phases using the aprotic solvent N- butylpyrrolidin-2-one (TamiSolveTM) in the binding buffer, with the protic solvent isopropanol (I PA) used as a positive control.
  • TamiSolveTM aprotic solvent N- butylpyrrolidin-2-one
  • I PA isopropanol
  • the nucleic acid may be a naturally occurring molecule, i.e. DNA or RNA but also include DNA/RNA hybrids where the DNA is in separate strands or in the same strand) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester linkage to the 5' position of the pentose of the next nucleotide.
  • Nucleic acids used in various embodiments may comprise chemically, enzymatically, or metabolically modified forms of nucleotides or combinations thereof, such as primers, probes, oligonucleotides or aptamers.
  • Exemplary types of DNA include synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g.
  • RNA examples include mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).
  • purification buffer means, unless the context requires otherwise, a buffer that is useful for precipitating a nucleic acid from solution to a solid support and/or a buffer that is useful for washing a nucleic acid bound to a solid support. Exemplary purification buffers may therefore also be considered binding buffers or washing buffers.
  • binding buffer means, unless the context requires otherwise, a buffer that is useful for precipitating a nucleic acid from solution to a solid support. Nucleic acids are often solvated in aqueous solutions, therefore typical binding buffers are miscible in aqueous solution, e.g. a binding buffer may be provided as an aqueous buffer.
  • Exemplary binding buffers of the present invention are aqueous buffers comprising a polar aprotic solvent.
  • wash buffer or “washing buffer” mean, unless the context requires otherwise, a buffer that is useful for washing a nucleic acid bound to a solid support. Binding buffers, in view of their function to precipitate nucleic acid from solution, may also be useful as wash buffers, as they should provide minimal loss of the nucleic acid from the solid support during washing. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the binding buffer, such as a 1.1 to 5 times dilution of a corresponding binding buffer.
  • elution buffer means, unless the context requires otherwise, a buffer that is useful for eluting (removing) a substance, e.g. nucleic acid or protein, from a solid support (to which the substance is bound).
  • the elution buffer to be used will depend on the substance to be eluted and the solid support.
  • a nucleic acid elution buffer may comprise water, Tris-HCI, EDTA or a combination thereof.
  • a protein elution buffer may comprise an aqueous salt solution, such as saline, a solution comprising glycine-HCI at a pH of aroundf 2.5-3.0, a solution comprising NH4OH and/or buffers suitable for protease digestion (e.g. trypsin digestion) of the eluted protein e.g. for subsequent mass spectrometry analysis.
  • aqueous salt solution such as saline
  • a solution comprising glycine-HCI at a pH of aroundf 2.5-3.0 a solution comprising NH4OH and/or buffers suitable for protease digestion (e.g. trypsin digestion) of the eluted protein e.g. for subsequent mass spectrometry analysis.
  • Targeted protein isolation is specific to a particular protein, or class of protein.
  • An example of targeted protein isolation is immunoprecipitation in which an antibody with a specific affinity for a protein of interest immobilized to a solid support (e.g. a bead) is contacted with the sample causing that protein to bind to the support (e.g. the bead).
  • solid support or “solid phase” means, unless otherwise stated, a material that is substantially insoluble in a selected solvent system (e.g. comprising an aqueous buffer), or which can be readily separated (e.g., by precipitation) from a selected solvent system in which it is soluble.
  • solid supports that are substantially insoluble in a selected solvent system e.g. comprising an aqueous buffer
  • Such solid supports are not limited to a specific type of support, and a large number of such solid supports are available and are known to one of ordinary skill in the art.
  • the surface of the solid support may comprise positively charged groups, for example one or more positively ionizable groups with a pK a of between about 5 and 8 (e.g. with a pK a of between about 6 and 7).
  • the surface of the solid support may comprise polyethylene imine groups, morpholine groups, alanine groups, polyhydroxy amine groups (such as Tris, Bis-Tris, and the like).
  • the hydrophilic surface of the solid support may comprise biological buffer covalently bound thereto; for example a biological buffer as described in US 6,914,137 B2, column 5, line 55 to column 6, line 59, covalently bound as described in said document at column 7, lines 29 to 64, the content of which is incorporated herein by reference in its entirety.
  • a solid support comprising surface silanol groups may be composed of comprise silica, which, at its surface, terminates in, inter alia, silanol groups ( -Si-OH).
  • Solid supports may comprise beads, for example monodisperse beads.
  • the beads may be monodisperse and I or magnetic and I or porous.
  • the (optionally monodisperse) beads may be magnetic and I or porous.
  • the beads may be monodisperse; as well as magnetic and I or porous.
  • the beads may be magnetic, e.g. the beads may be monodisperse and magnetic.
  • the magnetic beads may comprise microparticles or nanoparticles.
  • the magnetic beads may contain iron oxide.
  • magnetic nanoclusters as described in patent application No. GB2210796.5 (and patent publication No. WO 2024/018084 A2), which are hereby incorporated by reference, may be used.
  • the solid support comprising surface silanol groups may be selected from any commercially available solid support suitable for binding nucleic acid comprising surface silanol groups.
  • the solid support comprising surface silanol groups may be selected from DynabeadsTM MyOneTM Silane, SeraSil-Mag 400 or 700 (Cytiva), Silicon Hydroxyl Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), and MagneSilTM beads (available from Promega).
  • the solid support comprising surface carboxyl groups may be selected from any commercially available solid support suitable for binding nucleic acid comprising surface carboxyl groups.
  • the solid support comprising surface carboxyl groups may be selected from DynabeadsTM MyOneTM Carboxylic Acid, DynabeadsTM M- 270TM Carboxylic Acid (all available from Thermo Fisher Scientific), SeraMag SpeedBeadsTM carboxylate-modified (Cytiva), BioMagPlus COOHTM and ProMag 1 COOHTM (both Bangs Laboratories, INC Fishers), 4.4 pm fluorescent ferromagnetic beads or 2.0 pm ferromagnetic beads (both available from Spherotech INC Lake Forest, IL), 2 pm beads designated WHM-S001TM or 2 pm beads designated WHM-S002TM (both available from Creative Diagnostics, New York, NY), Carboxyl Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), Carboxyl Adembeads (available at different nm
  • the solid support used to immobilize the protein affinity reagent may be selected from any commercially available solid support suitable for binding protein.
  • the solid support used to immobilize the protein affinity reagent may be selected from e.g. DynabeadsTM MyOneTM Epoxy, DynabeadsTM M-270 Epoxy beads, DynabeadsTM MyOneTM Tosylactivated and DynabeadsTM Protein A, DynabeadsTM Protein G, DynabeadsTM Protein A/Protein G and DynabeadsTM M-280 Streptavidin, Dynabeads M- 270 Streptavidin, Dynabeads MyOne Streptavidin C1, Dynabeads MyOne Streptavidin T1, DynabeadsTM Sheep-Anti Mouse IgG, DynabeadsTM M-280 Sheep Anti-Mouse IgG, DynabeadsTM M-280 Sheep Anti-Rabbit IgG, DynabeadsTM Sheep Anti-
  • alcohol means, unless otherwise stated, a compound comprising a hydroxyl group, for example an alkane or alkene substituted by a hydroxyl group.
  • exemplary alcohols include Ci-Ce alcohol; such as methanol, ethanol, propanol, isopropanol, or butanol.
  • purification buffers, binding buffers, wash buffers and aqueous medium that comprise an aprotic solvent may not comprise alcohol.
  • glycol ether means, unless stated otherwise, a compound comprising a hydroxyl group and an ether group.
  • exemplary glycol ethers include, but are not limited to, a C2-C10 glycol ether, where the glycol ether can be an alkane or alkene substituted by a hydroxyl group and interrupted by 1, 2 or 3 ether linkages, where chemically possible.
  • Exemplary glycol ethers include 2-methoxyethanol, 2-ethoxyethanol, 2-propoxyethanol, 2- isopropoxyethanol, 2-butoxyethanol, 1-methoxy-2-propanol, 2-(2-methoxyethoxy)ethanol, and 2-(2-ethoxyethoxy)ethanol.
  • polyol means, unless otherwise stated, a compound comprising multiple (e.g. 2) hydroxyl groups.
  • exemplary polyols include, but are not limited to, a C2- C10 polyol, where the polyol can be an alkane or alkene substituted by at least two hydroxyl groups and optionally interrupted by 1, 2 or 3 ether linkages.
  • Particular examples of polyols include 2-methyl-1,3-propanediol, tripropylene glycol, and butanediol.
  • purification buffers, binding buffers, wash buffers and aqueous medium that comprise an aprotic solvent may not comprise polyol.
  • magnetic means responds to a magnetic field.
  • magnetic beads respond to a magnetic field.
  • Magnetic materials may be paramagnetic or superparamagnetic. When the magnetic material is paramagnetic, the magnetic properties are switched off when the magnetic field is removed. When the magnetic material is superparamagnetic, the magnetic material becomes saturated at relatively low magnetic fields and switching off of the magnetic properties with removal of the magnetic field is very rapid/instant.
  • Some magnetic material e.g. iron oxides, form superparamagnetic crystals when the size of the crystals is sufficiently small (e.g. below about 15 nm scale for iron oxides).
  • DNA deoxyribonucleic acid dPCR digital polymerase chain reaction dsDNA double Stranded DNA dsRNA double Stranded RNA
  • RTqPCR quantitative reverse transcription polymerase chain reaction SDS sodium dodecyl sulfate siRNA mall interfering RNA snRNA small nuclear RNA ssRNA single-stranded RNA
  • the sample is initially subject to protein separation (e.g. immunoprecipitation) to isolate any protein in the sample. Any isolated protein can then be subject to various downstream processes.
  • An aqueous nucleic acid purification buffer comprising a polar aprotic solvent and solid support (e.g. magnetic beads) comprising surface silanol groups are then added to the sample.
  • the formed DNA-bound solid support is then washed and eluted to form free DNA which can then be subject to various downstream processes.
  • Solid support e.g. magnetic beads
  • Solid support comprising surface carboxyl groups is then added to the sample.
  • the formed RNA-bound solid support is then removed, washed and eluted to form free RNA which can then be subject to various downstream processes.
  • Step 0 of Figure 2 Enrichment of the source of protein and/or nucleic acid to be isolated using techniques known in the art, e.g. circulating tumor cell (CTC) or exosome isolation using e.g. DynabeadsTM magnetic beads coupled to CTC or exosome specific antibodies, or SAX/Charge Switch beads for generic exosome enrichment.
  • CTC circulating tumor cell
  • DynabeadsTM magnetic beads coupled to CTC or exosome specific antibodies e.g. DynabeadsTM magnetic beads coupled to CTC or exosome specific antibodies, or SAX/Charge Switch beads for generic exosome enrichment.
  • Step 1 of Figure 2 Protein isolation from liquid biopsy sample matrix cleared of cells.
  • the protein may similarly be isolated from, for example, single cell or tumor-derived exosome lysates, or from any other source of protein and nucleic acids (e.g. plasma, serum, urine, etc.) suitable for immunoprecipitation of protein targets.
  • the protein isolation from the sample is provided by adding target-specific antibody coupled beads directly into the sample matrix. Move bead/protein complex to a fresh tube for further processing until the captured protein is ready for downstream readout (e.g. mass spectrometry, immunoassay, etc).
  • a non-targeted protein isolation may be performed.
  • Step 2 of Figure 2 DNA isolation by adding ProK, Lysis buffer, DynabeadsTM
  • MyOneTM Silane and the aprotic solvent to trigger DNA to bead binding. Move bead/DNA complex to a fresh tube for downstream processing.
  • Step 3 of Figure 2 RNA isolation by adding DynabeadsTM MyOneTM Carboxylic acid to the DNA-cleared lysate for RNA binding. Remove sample matrix, and continue processing.
  • Step 4 of Figure 2 RNA isolation by adding DynabeadsTM MyOneTM Carboxylic acid to the DNA-cleared lysate for RNA binding. Remove sample matrix, and continue processing.
  • Methods for isolating nucleic acids often involve exposing the nucleic acid to an aqueous solution, followed by precipitating the nucleic acid onto a solid support. Examples of such methods are provided in WO 2012/069660. An important component in such a method is the buffer used to precipitate the nucleic acid onto the solid support.
  • the nucleic acid purification buffer comprising a polar aprotic solvent utilised in the method of the present invention provides a number of advantages when compared to purification buffers used in the prior art, which are based on, e.g. alcohols and polyols.
  • the inventors have shown that purification buffers comprising a polar aprotic solvent may promote differential binding of DNA and RNA to solid supports (e.g. beads) functionalized with silanol and carboxyl groups, respectively, from the same sample, without the need to change the purification buffer. This greatly simplifies and reduces the complexity and cost of the total DNA, RNA and, optionally, protein sequential isolation workflow for multiomics readout.
  • the polar aprotic solvent is usually non-flammable and has low volatility, features that are not shared by common alcohols. This provides benefits such as improved safety for handling and shipping, e.g. allowing the buffer to be packaged and shipped in sealed containers and cartridges in ready to use format. The low volatility also provides for a reduced level of component concentration variation over time.
  • the polar aprotic solvents typically have relatively low viscosity (e.g. viscosity of less than 50 cP at 20 °C and 1 atm), which allows for a good level of accuracy and ease when measuring and dispensing the buffer.
  • the buffers may also provide other benefits, such as being relatively environmentally friendly (comprising components that are biodegradable and renewable), health and safety compliant, and being compatible with downstream nucleic acid processing/analysis procedures, such as amplification or mass spectrometry.
  • the polar aprotic solvent may be or comprise N-butylpyrrolidin-2-one (TamisolveTM):
  • the polar aprotic solvent may be or comprise dihydrolevoglucosenone (CyreneTM):
  • the polar aprotic solvent may be or comprise dipropylene glycol dimethyl ether (ProglydeTM):
  • Exemplary chaotropes include guanidinium salts (such as GuSCN and GuHCI), urea, and the like.
  • Surfactants may be selected from nonionic surfactants (such as TritonTM X-100, DDM, digitonin, TweenTM 20, TweenTM 80, EcosurfTM, Brij®, and the like), anionic surfactants (such as sodium dodecyl sulfate, deoxycholate, cholate, sarkosyl, and the like), and zwitterionic surfactants (such as CHAPS, Zwittergent® 3-14, and the like).
  • Buffering agents may be any suitable buffers that would provide buffering in the pH range of about 5 to about 9.
  • buffering agents include Tris, Trizma, Citrate, phosphate, Tricine, TAPS, PBS, acetate, borate, HEPES, Bicine, MOPS, CHES, carbonate, and the like, as well as mixtures thereof; and the buffer may also comprise other components such as polyethylene glycol (PEG), tetraethylene glycol (TEG), and linear polyacrylamide (LPA).
  • Enzymes are typically protein or other degrading enzymes, such as proteinase K or lysozyme.
  • Exemplary inorganic salts include metal halides, such as MgCh, NaCI, LiCI, and the like.
  • nucleic acid purification buffer and (nucleic acid) binding buffer may be used interchangeably.
  • the lysis or lysis/binding buffer typically comprises a chaotrope. Chaotropic agents denature macromolecules in the biological material, such as proteins and nucleic acids. Chaotropic agents also disrupt membrane lipids. Thus, the chaotropic agent in the lysis solution functions to reduce enzymatic activity and facilitate the induction of cell lysis. Any suitable chaotropic agent may be used in the lysis or lysis/binding buffer. Any suitable combination of chaotropic agents may be used in the lysis or lysis/binding buffer. For instance, in some embodiments, the chaotropic agent is selected from a guanidinium salt (e.g.
  • the chaotropic agent is selected from a guanidinium salt (e.g.
  • guanidinium isothiocyanate guanidinium isocyanate or guanidinium hydrochloride
  • lithium perchlorate lithium acetate, magnesium chloride, n-butanol, ethanol, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, thiocyanate salts, urea and a combination thereof.
  • the chaotropic agent or chaotrope may be present in the lysis or lysis/binding buffer at a concentration sufficient to denature macromolecules in the biological material and/or induce cell lysis.
  • the lysis or lysis/binding buffer contains a guanidinium salt at a concentration of at least about 2M, at least about 3M, such as at least about 3.5M or about 4M, e.g about 3-6M. In some embodiments, the lysis or lysis/binding buffer contains a guanidinium salt at a concentration of at least about 1M, at least about 1.5M, at least about 2M, at least about 3M, such as at least about 3.5M or about 4M, e.g. about 1-6M.
  • the lysis buffer contains guanidinium (iso)thiocyanate at a final concentration of about 1.0- 4.5M, 1.5- 3.5M, or 3.5-4.5M, or guanidinium hydrochloride at a concentration of about 3.5- 4.5M, e.g. about 4.0M.
  • the final concentrations during lysis and during binding steps differ due to addition of sample and trigger, respectively.
  • the lysis or lysis/binding buffer typically comprises a surfactant, for example a non-ionic surfactant or detergent, of which poly(oxyethylene)-containing surfactants may be mentioned.
  • the detergent in the lysis solution functions to disrupt cellular and organelle membranes, e.g. lyse cells and organelles, and to denature proteins in the biological material.
  • detergents function to facilitate the release of nucleic acids from cells and other entities, e.g. viruses, in the biological material.
  • Any suitable detergent may be used in the lysis or lysis/binding solution, e.g. a non-ionic detergent.
  • the detergent is selected from sodium lauroyl sarcosinate (sarkosyl), sodium dodecyl sulfate (SOS) polyoxyethylene-20-sorbitan monolaurate (Tween®- 20)TM, EcosurfTM, CHAPS, Brij®, Zwittergent® 3-14 and a combination thereof.
  • the detergent is selected from polyoxyethylene-20-sorbitan monolaurate (Tween®-20).
  • the detergent is present in the lysis solution at a concentration sufficient to disrupt cellular and organelle membranes, e.g. lyse cells and organelles, disrupt viral envelopes and/or capsids, and/or to denature proteins in the biological material.
  • the detergent is present at a concentration of about 0.5-5.0% w/v, e.g. about 0.75-4.5% w/v, about 1.0-4.0% w/v, about 1.5-3.0% 20 w/v, such as about 1.75-2.25% w/v, e.g. about 2.0%.
  • the detergent is sodium lauroyl sarcosinate (sarkosyl) or polyoxyethylene-20-sorbitan monolaurate (Tween®-20) at a concentration as defined above.
  • the lysis/binding buffer may be any buffer known for this purpose, e.g. with a high concentration of chaotrope (for example a guanidinium salt or urea) and surfactant, e.g. polyoxyethylene-20-sorbitan monolaurate (Tween®-20).
  • a preferred lysis buffer is a high detergent, guanidinium isothiocyanate (GTC)-containing buffer.
  • the lysis solution comprises EDTA at a concentration as defined above.
  • one or more other inhibitors of DNases and/or RNases known in the art may be present in the lysis/binding buffer.
  • a lysis or lysis/binding buffer may also contain one or more reducing agent.
  • the reducing agent functions to reduce disulfide bonds in proteins in the biological material.
  • Suitable reducing agents are well-known in the art and may be selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), p- mercaptoethanol (P-ME) and a combination thereof.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • P-ME p- mercaptoethanol
  • the use of TCEP may be particularly advantageous because it has high stability and activity at room temperature, thereby facilitating the production of a lysis buffer with improved activity that can be stored long term (e.g. useful for large scale production of a commercial product).
  • the reducing agent is TCEP.
  • the reducing agent when included in the lysis or lysis/binding buffer, may be present at a concentration sufficient to reduce disulfide bonds in proteins in the biological material.
  • the reducing agent may be present at a concentration of about 1-20 mM, such as about 2-19 mM, 3-18 mM, 4-17 mM or about 5-16 mM, e.g. about 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 mM, preferably about 10 mM.
  • a higher amount of reducing agent may be used, e.g. about 20-150 mM, such as about 25-125 mM or about 30-100 mM, e.g about 80 mM.
  • the lysis step may also use other components such as a protease, e.g. proteinase K.
  • a proteinase may enhance the efficiency of nucleic acid extraction.
  • the lysis may therefore be performed by combining a cell, a virus or another biological structure with a chaotrope (for example a guanidinium salt or urea), a surfactant (for example a non-ionic surfactant, e.g. a polyoxyethylenic surfactant) and a protease (such as a proteinase, for example proteinase K).
  • a protease such as a proteinase
  • a protease may be included in the lysis buffer.
  • a protease such as a proteinase
  • a protease may be added to the sample after it has been contacted with the lysis buffer but before it is contacted with the binding or purification buffer.
  • a protease (such as a proteinase) may be added contemporaneously when contacting the sample with a solid support (e.g. with a suspension of magnetic particles).
  • the lysis/ binding buffer is commonly supplemented with isopropanol e.g. 50% isopropanol, or it may be supplemented with ethanol, to facilitate capture of the nucleic acid on the solid support.
  • isopropanol e.g. 50% isopropanol
  • ethanol e.g. 50% ethanol
  • Point-of-care detection may be performed using a point-of-care instrument.
  • the point-of-care detection may comprise use of a cartridge or microfluidic chip.
  • the cartridge may comprise compartments containing one or more of the solid support, aqueous medium comprising a polar aprotic solvent (e.g. aqueous nucleic acid purification buffer as disclosed herein), wash buffer, and elution buffer.
  • the cartridge may also comprise the necessary microfluidics to transfer reagents between compartments to perform the methods of processing nucleic acids described herein.
  • Such cartridges may be typically shipped and stored in ready to use format, so it is advantageous if reagents (such as aqueous medium comprising a polar aprotic solvent and/or buffers) contained therein are not flammable.
  • An exemplary point-of-care-detection method may comprise introducing the sample to a cartridge or microfluidic chip, performing the following steps on the cartridge: exposing a sample comprising the nucleic acid to an aqueous medium comprising a polar aprotic solvent in the presence of a solid support; precipitating the nucleic acid to the solid support, thereby providing a nucleic acid bound solid support; washing the nucleic acid bound solid support with a wash buffer; and contacting the nucleic acid bound solid support with an elution buffer, thereby separating the nucleic acid from the solid support to provide a separated nucleic acid; and then subjecting the nucleic acid to one or more additional processes.
  • Examples of additional processes that may be relevant to point-of -care detection include but are not limited to detection, quantification, cloning, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridisation, cDNA synthesis, size separation, chromatography, and mass spectrometry.
  • the sample may be the supernatant of one or more targeted protein isolations and/or non-targeted protein isolation.
  • Kits of the invention may be used in both automated liquid handling, pipetting systems or in automated multi-purpose, high-throughput integrated laboratory systems that include downstream processing functionalities.
  • Exemplary automated protein and/or nucleic acid analysis platforms to be used with the present invention could be including, but not limited to, liquid handling and automation systems such as KingFisherTM Systems, DreamPrepTM NAP workstation (TECAN), FluentTM Automation Workstation (TECAN), Microlab Prep, NIMBUS, STAR or VANTAGE pipetting platforms (Hamilton Company).
  • compatible automated systems could provide downstream processing, such as genotyping or diagnostics testing, e.g. CORTM MX/PX or GX System (Beckton Dickinson), COBASTM 5800 system (Roche Diagnostics).
  • the automated nucleic acid analysis platform may be a point-of care assay instrument.
  • Point-of-care assay instruments may be adapted for detection of clinical markers and/or drugs, and/or pathogen detection, and/or biological warfare agent detection, and/or genetic disease detection.
  • the pathogen detection or biological warfare agent detection may comprise viral and/or bacterial and/or fungal protein and/or nucleic acid detection.
  • Exemplary point-of care assay instruments include ePlex® systems available from GenMark Diagnostics, Inc. of Carlsbad, CA, USA, and Solana® instruments available from Quidel of San Diego, CA, USA.
  • Currently available instruments also include cobas® 8000 modular analyzer series from Roche, the VIDAS® systems from bioMerieux or ARCHITECT analysers from Abbott.
  • Siemens, DiaSorin and Ortho Clinical Diagnostics provide multi target analyser systems as well.
  • KRYPTORTM analysers are marketed by Thermo Fisher. Further examples of point-of-care assay instruments are provided in US 9,752,182 B2 and US 2016/0016171 A1 , the content of which is incorporated by reference herein.
  • a method of sequentially isolating DNA and RNA from a sample comprising steps a) to d): a) contacting the sample with a solid support comprising surface silanol groups in the presence of an aqueous nucleic acid purification buffer to provide a DNA- bound solid support; b) removing the DNA-bound solid support from the sample; c) contacting the sample with a solid support comprising surface carboxyl groups in the presence of the aqueous nucleic acid purification buffer to provide an RNA-bound solid support; and d) removing the RNA-bound solid support from the fluid sample, wherein the aqueous nucleic acid purification buffer comprises a polar aprotic solvent.
  • polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine.
  • the affinity protein separation comprises: contacting the sample with a protein affinity reagent immobilized to a solid support to provide a protein-bound solid support; and removing the protein-bound solid support from the fluid sample.
  • the lysis step is performed before step a) or during step a); optionally wherein the lysis step is performed before affinity protein separation.
  • the method of clause 8 or 9, wherein the lysis step comprises adding a lysis buffer to the sample.
  • the method of any preceding clause further comprising contacting the sample with Proteinase K prior to step a), provided that said Proteinase K is not added until after the (optional) affinity protein separation.
  • the solid supports comprise (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.
  • step b) further comprises b2) washing the DNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer.
  • step b) further comprises b3) contacting the DNA-bound solid support with an elution buffer, thereby separating the DNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.
  • step d) further comprises d2) washing the RNA-bound solid support with a wash buffer, optionally wherein the wash buffer is or comprises the purification buffer.
  • step d) further comprises d3) contacting the RNA-bound solid support with an elution buffer, thereby separating the RNA its solid support, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA or a combination thereof.
  • the separated RNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and next-generation sequencing.
  • the polar aprotic solvent is present in an amount of at least about 2 %wt, optionally wherein the polar aprotic solvent is present in an amount of not more than about 80 %wt.
  • the purification buffer further comprises one or more of a chaotrope, a surfactant, a buffering agent, enzyme, inorganic salt, antifoaming agent or a combination thereof. 21.
  • the purification buffer is a binding buffer and/or a wash buffer, optionally wherein the wash buffer comprises a 1.1 to 5 times dilution of the binding buffer.
  • the sample is a biological sample
  • the biological sample is a fluid biological sample (e.g. a liquid biopsy); the biological sample is a cell based sample; the biological sample is single cell-based sample, CTC, exosomes, tissue biopsy material or FFPE; the biological sample comprise CTCs, the DNA comprise gDNA and the RNA comprise total RNA; or the biological sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.
  • the biological sample is a fluid biological sample (e.g. a liquid biopsy); the biological sample is a cell based sample; the biological sample is single cell-based sample, CTC, exosomes, tissue biopsy material or FFPE; the biological sample comprise CTCs, the DNA comprise gDNA and the RNA comprise total RNA; or the biological sample comprises exosomes, the DNA comprises cfDNA and the RNA comprises one or more of miRNA, mRNA, and snRNA.
  • the biological sample is
  • the polar aprotic solvent is selected from one or more of dihydrolevoglucosenone, N-butylpyrrolidin-2-one, dipropylene glycol dimethyl ether, and N-Formylmorpholine, optionally wherein the polar aprotic solvent is N- butylpyrrolidin-2-one.
  • kit of clause 26 or 27 further comprising a third solid support comprising an immobilized protein affinity reagent.
  • the kit of any one of clauses 26 to 29 further comprising Proteinase K. 31.
  • the kit of any one of clauses 26 to 30, wherein the first solid support, and/or second solid support, and/or (optional) third solid support comprises (optionally monodisperse) beads, further optionally wherein the (optionally monodisperse) beads are magnetic.
  • kit of any one of clauses 26 to 31 further comprising at least one wash buffer, optionally wherein the at least one wash buffer comprises the purification buffer.
  • kit of any one of clauses 26 to 32 further comprising an elution buffer, optionally wherein the elution buffer comprises water, Tris-HCI, EDTA, or a combination thereof.
  • Carboxylic acid magnetic beads DynabeadsTM MyOneTM Carboxylic Acid (Thermo Fisher Scientific).
  • Trigger aprotic solvent for nucleic acid purification buffer, as set out in Table 1 for embodiments. Isopropanol (I PA) was used as the alternative trigger for positive controls.
  • Wash buffer 2 70% ethanol in water. Note that similar results would be expected if the 70% ethanol was replaced with about 70% trigger.
  • LB Buffer comprising guanidine salt, ionic or non-ionic detergent (e.g. a Tween, such as Tween 20), pH 5-9.
  • ionic or non-ionic detergent e.g. a Tween, such as Tween 20
  • Magnetic separator DynaMagTM- 2 magnet, SKU 12321 D (Thermo Fisher Scientific). This device provides a rack for microtubes, with a magnet in its base to provide efficient separation of magnetic beads from supernatant.
  • Microtube heating block pre-heated to desired lysis and/or elution temperature
  • Microtube bench top centrifuge 1000 pL, 200 pL, and 100 pL pipettors.
  • Protein isolation (optional): an exemplary workflow for an immunoprecipitation from liquid biopsies or soluble protein is provided in steps a) to g): a) Dilute the sample in 0.5 vol in PBS or PBST (pH 7.4) containing 5mM EDTA or a HEPES (pH 7.4) buffer with a detergents like Tween-20, such as those provided by the DynabeadsTM Co-Immunoprecipitation Kit (Thermo Fisher Scientific catalogue number 14321 D) b) Add your sample containing the antigen (Ag) (typically 100-1,000 pl) to the tube containing Dynabeads coupled Antibodies and gently pipette to resuspend the Dynabeads.
  • g) Optionally elute the bound proteins in a desired volume of either 0.5M NH4OH with 0.5 mM EDTA (for Mass Spec analysis) or 200 mM Glycine (pH 2.8) for standard gel analysis.
  • Trigger was gently added to the lysate and mixed with a brief pulse-vortex followed by incubation of the tube on a rotator/roller at room temperature for 10 minutes. In a thermal mixer/shaker, 10 min. incubation at 1050 rpm would be sufficient.
  • Wash Buffer 1 first wash: After removing the tube from the magnet, 800pl Wash Buffer 1 were added and vortexed until the beads were fully resuspended. The tube was placed onto the DynaMagTM-2 magnet and beads were allowed to collect to the magnet for 1-2 minutes.
  • Wash Buffer 1 second wash a second wash was performed by repeating steps 9-10 using the same volume of Wash Buffer 1 as in step 9.
  • Wash Buffer 2 second wash Steps 13 and 14 were repeated (but without transferring the bead solution to a fresh tube).
  • the tube was placed on the DynaMagTM-2 magnet. Beads were allowed to collect to the magnet for 15 seconds.
  • the eluted DNA was transferred t to a fresh 1.5 mL tube and stored at the appropriate temperature (e.g. 4 °C to -80 °C).
  • Example 2 Wash buffer variation
  • the protocol for the sequential extraction of DNA and RNA as described in Example 1 was repeated (positive control and Examples 2a-f).
  • the trigger, wash buffer 1 and wash buffer 2 used in each repeat, as well as the DNA and RNA purification rates after the first and second elutions, respectively, are provided in Table 2, below. While this does not affect initial binding, final recovery can be influenced by the choice of wash buffer 1 and wash buffer 2.
  • a plasma sample was spiked with 500 pg/mL IL-6 protein and 5.000.000 x 10 6 copies/mL of 120bp DNA.
  • 200 pl plasma with IL-6 and 120bp DNA was used per extraction and pipetted into a KingFisher plate.
  • 20 pg DynabeadsTM MyOneTM Tosylactivated coupled with anti-IL-6 antibody were added, mixed slowly for 20 min, and placed on a magnet. The supernatant was aspirated and used for the isolation of nucleic acids.
  • the beads were transferred into 200 pL Assay Buffer (TBST-BSA) and resuspended. 100 pL were transferred into white flat-bottom 96-well plate.
  • acridinium ester (AE) labelled detection antibody 50 pL (0.1 pg/mL) acridinium ester (AE) labelled detection antibody were added and the plate was incubated on a shaker at 1000 rpm at 37 °C. Using a plate washer with a magnet the beads were washed 3x with 300 pL TBS. Finally, the beads were resuspended in 50 pL TBST and the signal was read on a Varioskan LUX using Flash. A standard curve for IL-6 was generated in parallel to quantify the amount of the precipitated protein.
  • control workflow used I PA as a trigger for precipitation to the beads while the experimental workflows (Examples 4a and 4b) used Tamisolve as a trigger, but with varied wash buffer 2.
  • the reagents used in each workflow, as well as the protein, DNA and RNA purification rates after each respective elution are provided in Table 4, below. Table 4: Protein, DNA and RNA purification rates
  • a polar aprotic solvent e.g.TamiSolveTM
  • an appropriate solid support e.g IL-6 antibody coupled to DynabeadsTM MyOneTM Tosylactivated
  • DynabeadsTM MyOneTM SILANE for DNA and DynabeadsTM MyOneTM Carboxylic Acid for RNA isolation

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Abstract

La présente invention concerne un procédé d'isolement successif de l'ADN et de l'ARN à partir d'un échantillon, le procédé comportant les étapes suivantes : mise en contact de l'échantillon avec un support solide comprenant des groupes silanol de surface en présence d'un tampon aqueux de purification d'acide nucléique pour obtenir un support solide lié à l'ADN; élimination du support solide lié à l'ADN de l'échantillon; mise en contact de l'échantillon avec un support solide comprenant des groupes carboxyle de surface en présence du tampon aqueux de purification d'acide nucléique pour obtenir un support solide lié à l'ARN; et élimination du support solide lié à l'ARN de l'échantillon fluide, le tampon aqueux de purification d'acide nucléique comportant un solvant aprotique polaire. La présente invention concerne également : un kit comportant ledit tampon de purification et des supports solides; l'utilisation dudit kit dans une plate-forme d'analyse automatisée d'acides nucléiques; et un appareil d'analyse d'acides nucléiques, contenant une plate-forme d'analyse automatisée d'acides nucléiques; ainsi que le kit susmentionné.
PCT/EP2024/078805 2023-10-12 2024-10-11 Isolement successif d'acides nucléiques Pending WO2025078670A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US20090143570A1 (en) * 2007-11-30 2009-06-04 Ge Healthcare Bio-Sciences Corp. Method for isolation of genomic dna, rna and proteins from a single sample
US20110172405A1 (en) * 2008-09-17 2011-07-14 Ge Healthcare Bio-Sciences Corp. Method for small rna isolation
WO2012069660A1 (fr) 2010-11-26 2012-05-31 Invitrogen Dynal As Utilisation de polyols pour traiter des acides nucléiques
US20160016171A1 (en) 2013-03-15 2016-01-21 Nanobiosym, Inc. Systems and Methods for Mobile Device Analysis of Nucleic Acids and Proteins
US20170198279A1 (en) * 2014-07-17 2017-07-13 Qiagen Gmbh Method for isolating rna with high yield
US9752182B2 (en) 2004-12-23 2017-09-05 Abbott Point Of Care Inc. Molecular diagnostics system and methods
WO2017211913A1 (fr) 2016-06-08 2017-12-14 Life Technologies As Support solide
CN116004613A (zh) * 2023-03-02 2023-04-25 上海美吉生物医药科技有限公司 一种基于磁珠法快速提取植物组织总rna的方法
WO2024018084A2 (fr) 2022-07-22 2024-01-25 Life Technologies As Particules

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US9752182B2 (en) 2004-12-23 2017-09-05 Abbott Point Of Care Inc. Molecular diagnostics system and methods
US20090143570A1 (en) * 2007-11-30 2009-06-04 Ge Healthcare Bio-Sciences Corp. Method for isolation of genomic dna, rna and proteins from a single sample
US20110172405A1 (en) * 2008-09-17 2011-07-14 Ge Healthcare Bio-Sciences Corp. Method for small rna isolation
WO2012069660A1 (fr) 2010-11-26 2012-05-31 Invitrogen Dynal As Utilisation de polyols pour traiter des acides nucléiques
US20160016171A1 (en) 2013-03-15 2016-01-21 Nanobiosym, Inc. Systems and Methods for Mobile Device Analysis of Nucleic Acids and Proteins
US20170198279A1 (en) * 2014-07-17 2017-07-13 Qiagen Gmbh Method for isolating rna with high yield
WO2017211913A1 (fr) 2016-06-08 2017-12-14 Life Technologies As Support solide
WO2024018084A2 (fr) 2022-07-22 2024-01-25 Life Technologies As Particules
CN116004613A (zh) * 2023-03-02 2023-04-25 上海美吉生物医药科技有限公司 一种基于磁珠法快速提取植物组织总rna的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections", INFECTION AND DRUG RESISTANCE, vol. 13, 2020, pages 455 - 483
"Viscosity of Liquids", 2012, OECD PUBLISHING, article "OECD Guidelines for the Testing of Chemicals, Section 1"
ZHANG Z C ET AL: "Surface Modification of Magnetic Silica Microspheres and Its Application to the Isolation of Plant Genomic Nucleic Acids", CHINESE JOURNAL OF ANALYTICAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 35, no. 1, 1 January 2007 (2007-01-01), pages 31 - 36, XP022857795, ISSN: 1872-2040, [retrieved on 20070101], DOI: 10.1016/S1872-2040(07)60024-3 *

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