WO2020069385A1 - Isolement de l'adn et de l'arn d'un seul et même échantillon - Google Patents

Isolement de l'adn et de l'arn d'un seul et même échantillon Download PDF

Info

Publication number
WO2020069385A1
WO2020069385A1 PCT/US2019/053565 US2019053565W WO2020069385A1 WO 2020069385 A1 WO2020069385 A1 WO 2020069385A1 US 2019053565 W US2019053565 W US 2019053565W WO 2020069385 A1 WO2020069385 A1 WO 2020069385A1
Authority
WO
WIPO (PCT)
Prior art keywords
magnetic particles
buffer
rna
dna
supernatant
Prior art date
Application number
PCT/US2019/053565
Other languages
English (en)
Inventor
Asmita PATEL
Antonia HUR
Han Wei
Lauren SAUNDERS
Original Assignee
Beckman Coulter, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beckman Coulter, Inc. filed Critical Beckman Coulter, Inc.
Publication of WO2020069385A1 publication Critical patent/WO2020069385A1/fr

Links

Classifications

    • 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

  • the ability to isolate DNA and RNA from a whole sample would greatly simplify methods of isolating nucleic acids from biological samples.
  • the instant disclosure addresses these and other needs.
  • This disclosure provides at least two methods for separately isolating DNA and RNA from a single sample.
  • the methods described herein rely on the selective binding of DNA and RNA to solid supports (e.g., magnetic particles) based on size (e.g., DNA and RNA molecules having greater than 10,000 bps).
  • the methods described herein take advantage of the ability of DNA to non- selectively bind to magnetic particles (e.g., paramagnetic particles comprising a functional group that non-selectively binds nucleic acids) under relatively mild precipitation conditions (e.g., using solution comprising relatively lower concentrations of crowding agents, salts, and C 2 -C9-alkanols), while RNA, which is typically smaller in size, remains in solution.
  • the RNA may also be induced to bind the beads by increasing the concentration of the crowding agent.
  • C2-C9 alkanols include (C 2 -C9)alkanols and mixtures of (C 2 - C9)alkanols.
  • “(C2-C9)alkanol” is meant an alcohol of the formula (C2-C9)alkyl-OH, wherein “alkyl” refers to straight chain and branched alkyl groups having from 2 to 9 carbon atoms such as ethyl, n-propyl, n-butyl, isopropyl, iso-butyl, sec-butyl, and t-butyl, such that the resulting (C2-C9)alkanol can be methanol, ethanol, n-propanol, n-butanol, isopropanol, iso-butanol, sec- butanol, t-butanol, and the like.
  • both RNA and DNA are bound to magnetic particles using conditions that cause both DNA and RNA to non-selectively bind the magnetic particles. Substantially all the RNA is then selectively eluted from the magnetic particles using precipitation conditions that can be relatively mild. The supernatant comprising the RNA is separated from the magnetic particles comprising at least substantially all the DNA. RNA is then isolated from the supernatant and the DNA isolated from the magnetic particles.
  • At least substantially all the DNA is bound to the magnetic particles using precipitation conditions that can be relatively mild and that cause the DNA to bind the magnetic particles, while substantially all the RNA remains in solution.
  • the supernatant comprising the RNA is separated from the magnetic particles comprising the DNA. RNA is then isolated from the supernatant and the DNA isolated from the magnetic particles.
  • FIG. 1 is a plot of nucleic acid yield from single blood sample (EDTA, heparin, PaxGene). The methods started with 400 uL PaxGene blood, 150 uL EDTA blood, or 200 uL heparin blood. DNA was quantified with picogreen, and RNA was quantified with Ribogreen by correcting for DNA fluorescence in each sample.
  • FIG. 2A is a plot showing the amount of DNA and RNA isolated from a 10 mg mouse liver tissue sample. DNA yield averaged 32 pg, RNA yield averaged 66 pg from three technical replicates.
  • FIG 2B is a size exclusion chromatogram of a mouse liver tissue sample showing DNA sizes averaged >60,000 bp.
  • FIG. 2C is a size exclusion chromatogram of a mouse liver tissue sample showing two prominent rRNA peaks were observed in the RNA fraction.
  • FIG. 3 A is a plot showing the amount of DNA and RNA isolated from a 10 mg mouse brain tissue sample. DNA yield averaged 5.5 pg and RNA yield averaged 1.8 pg from three biological replicates.
  • FIG 3B is a size exclusion chromatogram of a mouse liver tissue sample showing DNA sizes averaged 20,000 bp or larger.
  • FIG 3C is a size exclusion chromatogram of a mouse liver tissue sample showing RNA integrity number (RIN) values were 9.1 - 9.7, showing good RNA integrity.
  • FIG. 4 A is a plot showing the yield of DNA and RNA.
  • DNA and RNA were isolated from increasing amounts of cells. Both DNA and RNA yield increased linearly with cell input.
  • FIGS. 4B-1-4B-3 and FIGS. 4C-1-4C-3 are size exclusion chromatograms showing that the DNA integrity number (DIN) and RIN scores for DNA and RNA isolated from 50,000, 100,000 and 500,000 cells were consistent and above 9, indicating that DNA and RNA is intact. The concentration of nucleic acids from 10,000 was too low for an accurate measurement.
  • FIG. 5 is a plot of nucleic acid yield from nasal swabs from a human subject.
  • the instant disclosure relates generally to a method of isolating DNA and RNA from a biological sample.
  • the instant disclosure also includes kits for isolating DNA and RNA from a biological sample.
  • the methods for isolating DNA and RNA from a biological sample described herein comprise: a) contacting the DNA and RNA from the sample with a first buffer comprising magnetic particles and binding at least substantially all of the DNA to the magnetic particles, while at least substantially all of the RNA remains unbound; b) separating the magnetic particles of step a) from a first supernatant comprising the RNA; c) isolating the RNA from the first supernatant; and d) isolating the DNA from the magnetic particles.
  • the first buffer can be any suitable buffer, such as a Tris-HCl buffer.
  • the first buffer can be a bind buffer that promotes the binding of DNA and/or RNA molecules to the magnetic particles described herein.
  • Example of bind buffers include Agencourt AMPure XP available from Beckman Coulter, Inc., Brea, CA, such as a bind buffer comprising about 3% to about 7% PEG with about 0.35 M to about 0.7 M NaCl (final concentration).
  • the bind buffer can be a 0.5X bind buffer.
  • the first buffer can further comprise salts, such as any Group (I) or Group (II) salt, including NaCl and KC1.
  • the first buffer can further comprise a suitable amount of at least one crowding agent, such as those known in the art.
  • a lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze, among other things, the amount of DNA and RNA present.
  • a non-limiting example of a lysis buffer is a buffer comprising 0.8 N NaOH/8% SDS lysis buffer, which can be prepared by combining 160 mL of 2 N NaOH, 160 mL of 20% SDS, and 80 mL of distilled water.
  • the isolating of the RNA from the first supernatant can comprise: contacting the first supernatant with the first buffer comprising magnetic particles and binding the RNA to the magnetic particles; replacing the first buffer with a solution to release the RNA from the magnetic particles; and separating the magnetic particles from a second supernatant comprising the isolated RNA.
  • Suitable solutions to release the RNA from the magnetic particles can be at least one of nuclease-free water, buffer comprising Tris and EDTA, and buffered water.
  • the RNA released from the magnetic particles can optionally be treated with a DNase.
  • the isolating the DNA from the magnetic particles comprises: contacting the magnetic particles bound to the DNA with a solution to release the DNA from the magnetic particles; and separating the magnetic particles from a third supernatant comprising the isolated DNA.
  • Suitable solutions to release the DNA from the magnetic particles can be at least one of nuclease-free water, buffer comprising Tris and EDTA, and buffered water.
  • the magnetic particles bound to the DNA or the DNA released from the magnetic particles can optionally be treated with an RNase.
  • the methods encompassed by the instant disclosure also include a method of isolating DNA and RNA from a biological sample comprising cells, the method comprising: i) contacting the biological sample with a lysis buffer and releasing DNA and RNA from the cells; ii) contacting the DNA and RNA with a first buffer comprising magnetic particles, thereby binding the DNA and the RNA to the magnetic particles; iii) contacting the magnetic particles of step ii) with a second buffer releasing at least substantially all of the RNA from the magnetic particles while at least substantially all of the DNA remains bound to the magnetic particles; iv) separating the magnetic particles of step iii) from a first supernatant comprising the RNA; v) isolating the RNA from the first supernatant; and vi) isolating the DNA from the magnetic particles.
  • the isolating the RNA from the first supernatant can comprise contacting the first supernatant with the first buffer comprising magnetic particles and binding the RNA to the magnetic particles; replacing the buffer with a solution to release the RNA from the magnetic particles; and separating the magnetic particles from a second supernatant comprising the isolated RNA.
  • Suitable solutions to release the RNA from the magnetic particles can be at least one of nuclease-free water, buffer comprising Tris and EDTA, and buffered water.
  • the RNA released from the magnetic particles can optionally be treated with a DNase.
  • the isolating the DNA from the magnetic particles can comprise contacting the magnetic particles bound to the DNA with a solution to release the DNA from the magnetic particles; and
  • Suitable solutions to release the DNA from the magnetic particles can be at least one of nuclease-free water, buffer comprising Tris and EDTA, and buffered water.
  • the magnetic particles bound to the DNA or the DNA released from the magnetic particles can optionally be treated with an RNase.
  • the term“crowding agent” generally refers to any material that allows for, enhances, or facilitates molecular crowding. Without wishing to be bound by any specific theory, it is believed that a crowding agent allows for molecular crowding and reduces the amount of water that comes into contact with the DNA and RNA molecules in a solution, allowing components of the solution to come into closer contact with one another. For example, DNA and/or RNA molecules can condense more quickly in the presence of the crowding agent, which facilitates methods described herein for separating DNA from RNA.
  • PEG polyethylene glycol
  • Ficoll such as Ficoll 70
  • dextran such as dextran 70
  • PEG polyethylene glycol
  • Any suitable size of PEG can be used, e.g., ranging from about PEG-200 (e.g., PEG-4000, PEG- 6000, or PEG-8000) to about PEG-20,000, or even higher.
  • the amount of the crowding agent used will be determined by the molecular weight of the crowding agent.
  • a concentration of about 5% (weight/volume) can be used.
  • the amount of crowding agent can range, e.g., from about 3% to about 7% or higher (e.g., 8%).
  • the first buffer can comprise components in addition to salts.
  • the first buffer can comprise magnetic particles.
  • the magnetic particles can be present in the buffer in any suitable amount, such as 1% (w/v) magnetic particles.
  • the magnetic particles can be any suitable magnetic particles.
  • the magnetic particles can be ferrimagnetic particles, ferromagnetic particles, paramagnetic particles, superparamagnetic particles, or mixtures of various classes of magnetic particles can also be used. Therefore, any specific recitation of a magnetic particle can be equally applied to a ferrimagnetic particle, ferromagnetic particle, paramagnetic particle, superparamagnetic particle, or mixtures thereof.
  • Suitable magnetic particles for use in the methods described herein include, but are not limited to AMPure XP beads available from Beckman Coulter, Inc., Brea, CA. Suitable magnetic particles also include those described in U.S. Patent Nos. 5,705,628; 5,898,071; and 6,534,262, and in PCT/US2019/042628, filed July 19, 2019, all of which are incorporated by reference as if fully set forth herein.
  • ferrimagnetic particles refers to particles comprising a ferrimagnetic material. Ferrimagnetic particles can respond to an external magnetic field (e.g., a changing magnetic field), but can demagnetize when the external magnetic field is removed. Thus, the ferrimagnetic particles are efficiently mixed through a sample by external magnetic fields as well as efficiently separated from a sample using a magnet or electromagnet, but can remain suspended without magnetically induced aggregation occurring.
  • an external magnetic field e.g., a changing magnetic field
  • the magnetic particles described herein are sufficiently responsive to magnetic fields such that they can be efficiently moved through a sample.
  • the range of the field intensity could be the same range as any electromagnet as long as it is able to move the particles.
  • the magnetic field has an intensity of between about 1 OmT and about 100 mT, between about 20 mT and about 80 mT, and between about 30 mT and about 50 mT.
  • more powerful electromagnets can be used to mix less responsive microparticles.
  • the magnetic field can be focused into the sample as much as possible.
  • the electromagnets can be as close to the sample as possible since the strength of the magnetic field decreases as the square of the distance.
  • the magnetic particles can be a variety of shapes, which can be regular or irregular; In some examples, the shape maximizes the surface areas of the particles.
  • the magnetic particles can be spherical, bar shaped, elliptical, or any other suitable shape.
  • the magnetic particles can be a variety of densities, which can be determined by the composition of the core. In some examples, the density of the magnetic particles can be adjusted with a coating, as described herein.
  • the magnetic particles have sufficient surface area to permit efficient binding of a target analyte and are further characterized by having surfaces which are capable of reversibly or irreversibly binding the target analyte (e.g., biological molecules, such as DNA and RNA).
  • a surface area of the magnetic particles can be in a range of from about 0.1 m 2 /g to about 500 m 2 /g, about 50 m 2 /g to about 200 m 2 /g, or about 150 m 2 /g to about 175 m 2 /g.
  • Suitable magnetic particles can be of a size that their separation from solution is not difficult, for example by magnetic means or by filtration. In addition, magnetic particles should not be so large that their surface area is minimized or that they are not suitable for nanoscale to microscale manipulation.
  • Suitable sizes range from about 1 nm mean diameter to about 1 mm mean diameter, about 5 nm to about 50 pm, or between about 100 nm and about 100 pm. A suitable is between about 1 pm and about 10 pm.
  • the magnetic particles can be nanoparticles (e.g., particles having a mean diameter less than 1 pm, but greater than 1 nm).
  • the magnetic particles can be microparticles (e.g., particles having a mean diameter greater than 1 pm, but less than 100 pm).
  • larger magnetic particles that is about 1 mm in size are useful in cellular fractionation, tissue digestion, liquid mixing, and the like.
  • the magnetic particles can be substantially solid or can have some degree of porosity. Where the magnetic particles do include some degree of porosity, a pore size of the individual pores can be in a range of from about 5 A to about 1000 A, about 50 A to about 500 A. At least a plurality of the pores can be through pores (e.g., extending fully between opposed surfaces).
  • the pore sizes or total porosity of the magnetic particles can be determined according to many suitable methods. For example, the bulk volume of an ideal (e.g., non-porous) magnetic particle can be determined and then the volume of the actual porous skeletal material can be determined. The porosity is then calculated by subtracting the volume of the actual porous skeletal material from the ideal magnetic particle.
  • the porosity of the magnetic particle or individual pore size can also be determined through optical measurements using a microscope and processing the images to measure the individual pores.
  • the magnetic particles described herein can include several different materials.
  • the total magnetic content of the magnetic particles can constitute at least 50 wt% of the magnetic particle, at least 70 wt% of the magnetic particle, or even 100 wt% of the magnetic particle.
  • the magnetic particles can include any of those described herein.
  • the non-magnetic material constituting the balance of the magnetic particles can include any of the coating materials described herein, for example.
  • Non-magnetic material can be used as a coating to encapsulate the magnetic portion of the magnetic particle, they can also be used as a functional component to interact with and bind an analyte of interest.
  • Non-magnetic material can also act a as filler component.
  • the magnetic strength of the magnetic particles can be greater than or equal to about 20 emu/g, about 25 emu/g, about 30 emu/g, about 35 emu/g, about 40 emu/g, about 45 emu/g, about 50 emu/g, about 75 emu/g, about 100 emu/g, about 150 emu/g, about 175 emu/g, about 200 emu/g, about 225 emu/g, about 250 emu/g, in a range of from about 20 emu/g to about 250 emu/g, or about 35 emu/g to about 100 emu/g.
  • This value can be considered to be the maximum field strength of the particle, which is a measure of the magnetic strength generated by the particle upon exposure to a magnetic field.
  • the permeability of the magnetic particle should be sufficient to generate an induced magnetic field greater than or equal to about 10 emu/g, 15 emu/g, 20 emu/g, about 25 emu/g, about 30 emu/g, about 35 emu/g, about 40 emu/g, about 45 emu/g, about 50 emu/g, about 75 emu/g, about 100 emu/g, about 150 emu/g, about 175 emu/g, about 200 emu/g, about 225 emu/g, about 250 emu/g, in a range of from about 10 emu/g to about 250 emu/g, or about 35 emu/g to about 100 emu/
  • the magnetic field to which the magnetic particles are exposed can have a strength of about 700 Oersted to about 800 Oersted, about 725 Oersted to about 775 Oersted, less than, equal to, or greater than about 700 Oersted, 725, 750, 775, or about 800 Oersted.
  • the remanence of the magnetic materials can be in a range of from about 0 emu/g to about 30 emu/g, about 0 emu/g to about 10 emu/g, about 1 emu/g to about 8 emu/g, about 3 emu/g to about 5 emu/g, less than, equal to, or greater than about 0 emu/g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 emu/g.
  • the magnetic components of the particles can be magnetic nanoparticles, magnetic sub-micrometer particles, or magnetic micrometer particles.
  • the magnetic particles described herein can have many different structures.
  • the magnetic particles can be magnetic nanoparticles incorporated in polymer matrix or silica matrix, magnetic beads encapsulated in silica shell or polymer shell, magnetic nanoparticles or magnetic beads functionalized with organic ligands, bare magnetic nanoparticles or beads.
  • the shell can include a coating as described herein.
  • the magnetic particles of the disclosure can comprise a magnetic core, surrounded by a coating.
  • the magnetic particles are coated with one or more layers of a non-magnetic material.
  • the use of coated magnetic particles, having no exposed iron, on their surfaces, can eliminate the possibility of iron interfering with certain downstream manipulations of the sample.
  • the coating can be, for example, a polymer layer, or a silica layer.
  • Example polymer layers can include polyethylene, polystyrene, poly methyl methacrylate, polyvinyl alcohol, or any other suitable polymer.
  • Example silica layers can include silicon dioxide, borosilicate, soda lime, barium titanate, and other types of glass.
  • the polymer or silica layer can be for adjusting the density of the magnetic particles.
  • the polymer or silica layer can adjust the density of the magnetic particles to be close to the density of the sample, for example, an aqueous sample (e.g., approximately 1 g/cm 3 ).
  • other types of coating can include metal plating such as aluminum, gold, zinc oxide, or any of the other coatings mentioned herein, etc.
  • any of the coatings described herein can have a fluorescent or colored dye included.
  • the coating can also comprise a ligand such as capture reagent or a functional group, including those mentioned herein, for selectively or non-selectively binding target analytes.
  • the functional group can be for adsorbing biomolecules, such as nucleic acids, which can non-sequence-specifically and reversibly bind to the functional group coating the magnetic particles.
  • the polynucleotides can be DNA, RNA, or polyamide nucleic acids (PNAs).
  • the functional group is a carboxyl group.
  • any of the coatings described herein can be functionalized with surface chemicals as described herein, for example, with carbolic acid, streptavidin, amine, hydrazide, silanol, azide. And those can be further functionalized with biological molecules such as antibodies, enzymes, DNA or RNA fragments, catalysts, etc.
  • the coating can comprise a capture reagent.
  • the capture reagent can be for capturing an analyte in a sample.
  • the surface of the magnetic particles can be coated with a capture reagent that is a suitable ligand or receptor (e.g., antibodies, lectins, oligonucleotides, other affinity groups, or any of the other capture reagents mentioned herein), which can selectively bind a target analyte or a group of analytes in a mixture.
  • the capture reagent can be an antibody.
  • capture reagents can be used for this purpose, e.g. aptamers, nanoparticles, binding proteins, and the like.
  • the capture reagent can be designed to capture a specific analyte or a specific panel of analytes, e.g., drug panel or endocrine panel, etc.
  • the ligand can include an enzyme.
  • the enzyme can be linked to the coating in order to selectively interact with a substrate of that enzyme. Upon interacting with the substrate, the enzyme can function to degrade or digest the substrate. This can lead to generation of a substance of interest through enzyme’s action or to remove a substrate from a sample.
  • the enzyme can be trypsin.
  • some examples can include multiple layers of coatings.
  • some examples can include a base metal coating with a polymer coating or functional group disposed thereon.
  • a layer of coating can function to sufficiently hold an external coating to the magnetic particle.
  • the magnetic particles can be manufactured using any suitable method of manufacturing nanoscale to microscale magnetic particles.
  • U.S. Patent No. 5,648, 124 discloses a process for preparing magnetically responsive microparticles, and is hereby incorporated by reference herein in its entirety.
  • the magnetic particles can be manufactured using any suitable magnetic material, as described herein.
  • a magnetic particle can be manufactured by first adding magnetic nanoparticles to a chemical bath.
  • the nanoparticles can be encapsulated in an inorganic silica matrix, thus producing a microparticle that contains many magnetic particles. Sonication can then be used to help produce these particles in a monodispersed fashion.
  • a silica matrix is mentioned above, it is also possible for individual magnetic nanoparticles or microparticles to be encapsulated in other inorganic or organic materials.
  • the magnetic nanoparticles can be encapsulated in Si0 2 , Ti0 2 , Zn0 2 , Al 2 0 3 , Ce0 2 , or any suitable ceramic material.
  • the magnetic nanoparticles can be encapsulated in an organic material such as polyacrylic acid (PAA), poly(methyl acrylate) (PMA), polystyrene (PS), divinylbenzene (DVB), polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA).
  • PAA polyacrylic acid
  • PMA poly(methyl acrylate)
  • PS polystyrene
  • DVB divinylbenzene
  • PVP polyvinylpyrrolidone
  • PVA polyvinyl alcohol
  • a ferromagnetic material can be used to manufacture magnetic particles.
  • the magnetic properties can be altered by changing the structure of the ferromagnetic material.
  • Hematite Fe?0, is naturally ferromagnetic when allowed to crystalize in its pure form. However, if impurities like nickel and zinc are added, then the nickel and zinc can take the place of some of the iron in the crystalline structure, thus turning the naturally ferromagnetic material into a magnetic particle.
  • ferromagnetic hematite can be ground down to less than 50 nm in size such that each particle contains a single magnetic domain. In this form, the particle can be a superparamagnetic particle.
  • An exemplary magnetic particle can be made from magnetic magnetite nanoparticles 50-100 nm in size joined together in silica or polymer. These nanoparticles are too large to be superparamagnetic.
  • a sample used in the present disclosure can be a fluid sample and can be, for example, a biological sample.
  • biological samples can comprise biological fluids and may include, but are not limited to, blood, plasma, serum, or other bodily fluids or excretions, such as but not limited to saliva, urine, cerebrospinal fluid, lacrimal fluid, perspiration, gastrointestinal fluid, amniotic fluid, mucosal fluid, pleural fluid, sebaceous oil, exhaled breath, and the like.
  • the biological sample can be bodily fluid or a tissue sample.
  • Biological samples can also be obtained from swabs used to collect animal cells, such as at least one of forensic, anal, vaginal, oral, buccal, aural, and nasal swabs.
  • Biological samples also include at least one of animal (e.g., mammalian and insect) tissue, animal (e.g., mammalian and insect) cells, bacteria, fungi, and yeast.
  • animal samples can be lysed samples, where, e.g., animal cells have been lysed using a lysing buffer, such as those described herein.
  • Appropriate biological samples may also include lysates prepared from cells obtained from either mammalian tissue, cell culture, or body fluids, nucleic acid samples eluted from agarose or polyacrylamide gels, solutions containing multiple species of DNA molecules resulting either from a polymerase chain reaction (PCR) amplification or from a DNA size selection procedure and solutions resulting from a post-sequencing reaction.
  • Suitable samples can be mixtures of biomolecules (e.g. proteins, polysaccharides, lipids, low molecular weight enzyme inhibitors, oligonucleotides, primers, templates) and other substances such as agarose, polyacrylamide, trace metals and organic solvents, from which the target nucleic acid molecule can be isolated.
  • the terms“selective” and“selectively” refer to the ability to isolate a particular biological molecule species such as a DNA molecule or molecules, on the basis of particular property, such as molecular size, from a combination which includes or is a mixture of species of molecules, such as a host cell lysate and other host cell components.
  • the selective isolation of a particular species is accomplished through the use of an appropriate crowding agent (e.g., polyalkylene glycol) to result in the precipitation and facilitated adsorption of a particular DNA species (e.g., characterized on the basis of size) to the surfaces of magnetic particles of the disclosure.
  • an appropriate crowding agent e.g., polyalkylene glycol
  • the term“at least substantially all” means that at least about
  • RNA or DNA nucleic acid
  • kits for isolating DNA and RNA from a biological sample comprising: a first buffer comprising magnetic particles; optionally a lysis buffer for releasing DNA and RNA from cells in the biological sample; and optionally one or more swabs for collecting the biological sample.
  • Lysis buffer Guanidine thiocyanate 3.1M; Triton X100 2% (V/V); Na citrate 400Mm; Tris Borate-EDTA 2X; Proteinase K.
  • this buffer generally comprises a crowding agent and a salt.
  • a specific, non-limiting example of a bind buffer comprises PEG 8000 10% (crowding agent); NaCl 0.875M (a salt); Tris-HCL pH:8 25 Mm; Beads: 1% (w/v).
  • IX Bind Buffer PEG 8000 21.3%; NaCl 1.75M; Tris-HCL pH:8 50 Mm; Beads: 2% (w/v).
  • Rebind Buffer this buffer generally comprises a crowding agent, a C2-C9- alkanol such as isopropanol or ethanol, and a salt. And as the name suggests, the rebind buffer promotes rebinding of RNA to the beads described herein.
  • a specific, non-limiting example of a rebind buffer comprises about 18% to about 22% PEG with about 1.75 M to about 2.26 M NaCl, such as PEG 8000 21.3%; NaCl 1.75M; Tris-HCL pH:8 50 Mm.
  • wash Buffer this buffer generally comprises at least one of a crowding agent or a C 2 - Cs-alkanol such as isopropanol or ethanol, with other components being optional.
  • the wash buffer is a buffer that would leave DNA and/or RNA present in a sample bound to the magnetic beads described herein and would allow for the removal of impurities present in a sample.
  • a specific, non-limiting example of a wash buffer comprises about 5% to about 22% PEG with about 0.4 M to about 2.26 M NaCl or about 70% or higher (e.g., about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher or about 95% ethanol, such as from about 70% to about 85%) ethanol and about 40% or higher (e.g., about 45% or higher, about 50% or higher, about 55% or higher, about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher or about 85% or higher) isopropanol will work.
  • a specific, non limiting example of a wash buffer is PEG 8000 12.5%; NaCl 1M; Tris-HCL pH:8 50 Mm.
  • lysis buffer Proteainase K, and 10 mg tissue were combined, and the tissue was homogenized. The sample was then incubated at 37°C for 25 minutes. After incubation, the homogenized tissue was centrifuged at 12000 rpm for 1 minute and the supernatant was transferred to a fresh tube. Bind buffer containing magnetic beads was added in sufficient volume (0.5x) to bind the DNA to the beads but not the RNA. The beads were pelleted on a magnet, and the RNA supernatant was removed.
  • RNA supernatant was then combined with lx bind buffer to bind the RNA to a fresh set of beads. These beads were then pelleted and the supernatant was removed. The beads were washed with 80% ethanol. Beads were then resuspended in a DNase solution to remove any contaminating DNA and were incubated at 37°C for 20 minutes. Rebind buffer was then added to rebind the RNA to the beads. The beads were pelleted and the supernatant was removed. The beads were then washed with 80% ethanol and finally the RNA was eluted in nuclease- free water.
  • the beads with bound DNA were resuspended in wash buffer containing RNase and incubated at room temperature for 5 minutes. The beads were pelleted on a magnet, then washed with 70% ethanol. The DNA was then eluted in nuclease-free water.
  • Example 1 The protocol from Example 1 was followed, except the samples were mammalian cells. Different amounts of cells were tested and no homogenization was done.
  • Example 1 The protocol from Example 1 was followed, except nasal swabs were used as the sample and DTT was added to the lysis buffer.
  • RNA samples e.g., PaxGene blood, which can be used as is; heparin blood and EDTA blood need to be mixed with detergent buffer (200 pL of each for heparin, 150 pL EDTA blood with 250 pL detergent buffer)).
  • detergent buffer 200 pL of each for heparin, 150 pL EDTA blood with 250 pL detergent buffer
  • 400 pL of blood sample are mixed with 300 pL RNAdvance Blood lysis buffer and 20 pL RNAdvance Blood PK.
  • the samples were incubated at 55°C for 25 min.
  • the samples are then mixed with 1 mL 1 X Bind buffer and incubated at room temperature for 5 minutes. This will bind both RNA and DNA.
  • RNA and DNA are separated on the magnet and supernatant is discarded. Samples are washed with Wash Buffer, then washed with 70% ethanol two times. Separation of RNA and DNA does not work with dirty initial blood samples.
  • RNA supernatant is mixed with 1 X bind buffer and isopropanol in the proportions listed in the RNAdvance Blood protocol. Beads are separated and supernatant is removed. DNase is added to samples (if needed). Samples are washed with 70% ethanol and eluted in water DNA pellet is washed with 100 pL wash buffer and 5 pL RNase (if needed). DNA is washed with 70% ethanol and eluted in water.

Abstract

L'invention concerne divers procédés d'isolement de l'ADN et de l'ARN d'un échantillon biologique.
PCT/US2019/053565 2018-09-28 2019-09-27 Isolement de l'adn et de l'arn d'un seul et même échantillon WO2020069385A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862738829P 2018-09-28 2018-09-28
US62/738,829 2018-09-28

Publications (1)

Publication Number Publication Date
WO2020069385A1 true WO2020069385A1 (fr) 2020-04-02

Family

ID=68296669

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/053565 WO2020069385A1 (fr) 2018-09-28 2019-09-27 Isolement de l'adn et de l'arn d'un seul et même échantillon

Country Status (1)

Country Link
WO (1) WO2020069385A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023198909A1 (fr) * 2022-04-15 2023-10-19 Quantoom Biosciences S.A. Procédés de séparation et/ou de purification d'acides nucléiques

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648124A (en) 1993-07-09 1997-07-15 Seradyn, Inc. Process for preparing magnetically responsive microparticles
US5705628A (en) 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US6534262B1 (en) 1998-05-14 2003-03-18 Whitehead Institute For Biomedical Research Solid phase technique for selectively isolating nucleic acids
US20060078923A1 (en) * 2003-04-02 2006-04-13 Mckernan Kevin Method for isolating nucleic acids
US20110172409A1 (en) * 2009-10-19 2011-07-14 Zhiqiang Han Method for nucleic acid isolation by solid phase reversible binding of nucleic acids
WO2012002887A1 (fr) * 2010-06-29 2012-01-05 Sjoeblom Tobias Procédé et kit d'isolation séquentielle d'espèces de nucléotide provenant d'un échantillon

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648124A (en) 1993-07-09 1997-07-15 Seradyn, Inc. Process for preparing magnetically responsive microparticles
US5705628A (en) 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US5898071A (en) 1994-09-20 1999-04-27 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US6534262B1 (en) 1998-05-14 2003-03-18 Whitehead Institute For Biomedical Research Solid phase technique for selectively isolating nucleic acids
US20060078923A1 (en) * 2003-04-02 2006-04-13 Mckernan Kevin Method for isolating nucleic acids
US20110172409A1 (en) * 2009-10-19 2011-07-14 Zhiqiang Han Method for nucleic acid isolation by solid phase reversible binding of nucleic acids
WO2012002887A1 (fr) * 2010-06-29 2012-01-05 Sjoeblom Tobias Procédé et kit d'isolation séquentielle d'espèces de nucléotide provenant d'un échantillon

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOHUSLAV RITTICH ET AL: "SPE and purification of DNA using magnetic particles : Sample Preparation", JOURNAL OF SEPARATION SCIENCE., vol. 36, no. 15, 9 July 2013 (2013-07-09), DE, pages 2472 - 2485, XP055561883, ISSN: 1615-9306, DOI: 10.1002/jssc.201300331 *
MAGORZATA A WITEK ET AL: "96-Well Polycarbonate-Based Microfluidic Titer Plate for High-Throughput Purification of DNA and RNA", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 80, no. 9, 1 May 2008 (2008-05-01), pages 3483 - 3491, XP002693159, ISSN: 0003-2700, [retrieved on 20080321], DOI: 10.1021/AC8002352 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023198909A1 (fr) * 2022-04-15 2023-10-19 Quantoom Biosciences S.A. Procédés de séparation et/ou de purification d'acides nucléiques

Similar Documents

Publication Publication Date Title
JP3253638B2 (ja) シリカ磁気粒子を使用する生物学的目標物質の分離法
US8569477B2 (en) Method for isolating nucleic acids comprising the use of ethylene glycol multimers
US20070026435A1 (en) Hydroxysilane functionalized magnetic particles and nucleic acid separation method
JP4594467B2 (ja) 常磁性粒子を用いた核酸の精製とその操作方法
EP2066709B1 (fr) Particules et leur utilisation dans un procédé d'isolement d'acide nucléique ou de phosphoprotéines
US20060024701A1 (en) Methods and reagents for the isolation of nucleic acids
DE60122138T2 (de) Isolierung von nukleinsäuren
US20200024592A1 (en) Highly active silica magnetic nanoparticles for purifying biomaterial and preparation method thereof
US8288169B2 (en) Surface mediated self-assembly of nanoparticles
US20030235839A1 (en) Solid phase technique for selectively isolating nucleic acids
US20070054285A1 (en) Method for isolating nucleic acids
JP6684868B2 (ja) 核酸の精製のための１工程法
CA2622037A1 (fr) Procede pour l'isolement d'acides nucleiques
JP2002512252A (ja) 核酸の精製における内毒素の低減
DE10201084A1 (de) Siliziumhaltige Magnetpartikel, Verfahren zu deren Herstellung und Verwendung der Partikel
WO2020069385A1 (fr) Isolement de l'adn et de l'arn d'un seul et même échantillon
US7332352B2 (en) Separation particles
JP2023536601A (ja) 分析物検出のためのサンプルのプール方法
WO2022183205A1 (fr) Particules magnétiques à sédimentation rapide et leurs applications
DE19549875B4 (de) Verwendung von magnetischen Partikeln zur Isolierung von Nukleinsäuren
MXPA98007681A (en) Methods of isolating biological target materials using silica magnetic particles

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19790935

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19790935

Country of ref document: EP

Kind code of ref document: A1