EP3022302A1 - Verfahren zur extraktion und reinigung von nukleinsäuren und verwendete puffer - Google Patents

Verfahren zur extraktion und reinigung von nukleinsäuren und verwendete puffer

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Publication number
EP3022302A1
EP3022302A1 EP14747318.5A EP14747318A EP3022302A1 EP 3022302 A1 EP3022302 A1 EP 3022302A1 EP 14747318 A EP14747318 A EP 14747318A EP 3022302 A1 EP3022302 A1 EP 3022302A1
Authority
EP
European Patent Office
Prior art keywords
buffer
sample
glycerol
washing
surfactant
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP14747318.5A
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English (en)
French (fr)
Inventor
Justine LEMONNIER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Filing date
Publication date
Application filed by Commissariat a lEnergie Atomique et aux Energies Alternatives CEA filed Critical Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Publication of EP3022302A1 publication Critical patent/EP3022302A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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 present invention belongs to the field of biology and, more particularly, to the field of molecular biology.
  • the present invention provides a method for extracting and purifying nucleic acids for one or more subsequent analysis (s) of the amplification or sequencing type.
  • the method according to the invention made from a sample containing nucleic acids uses buffers containing glycerol and particles or beads, magnetic or magnetizable.
  • the present invention also relates to the buffers used during this process and in particular the washing and optionally lysis buffers.
  • the lysis step of the methods of the prior art must make it possible to extract the nucleic acids from the material containing them while preserving their integrity.
  • the lysis step should be mild enough to preserve the nucleic acids while inhibiting or disabling the enzymes that destroy these nucleic acids, i.e. the nucleases.
  • the lysis can be mechanical, chemical and / or enzymatic.
  • Steps (ii) and (iii) correspond, strictly speaking, to the purification of the nucleic acids. These steps can implement one or more techniques selected from
  • a solvent extraction such as phenol-chloroform extraction
  • a precipitation such as an alcoholic precipitation or a precipitation with high saline concentrations
  • chromatographic technique such as gel filtration, ion exchange chromatography, adsorption chromatography or affinity chromatography and
  • centrifugation or ultracentrifugation such as centrifugation on cesium chloride.
  • This specific adsorption is linked to the charge of the beads such as positively or negatively charged beads or the presence, at the surface of the beads, of groups to which the nucleic acids bind such as anti-DNA antibodies or oligonucleotides such as poly (Thymine) oligonucleotides (or oligo (dT)).
  • US Patent 6,855,499 to Cortex Biochem Inc. aims to provide a simple method for isolating and purifying nucleic acids such as DNA (for "Deoxyribonucleic Acid”), RNA (for "RiboNucleic Acid”) or ANP (for "Peptide Nucleic Acid”) from various samples such as blood, milk, seminal fluid, tissue or bacterial cell lysates without centrifugation, alcohol and without preliminary preparation of the buffers.
  • the technical solution proposed in this patent consists in using cellulose particles which are in the form of magnetic or paramagnetic particles, coated with cellulose or one of its derivatives.
  • the bonding buffer described is composed of polyethylene glycol with a molar mass 8000 g / mol (or PEG 8000) at 10% and 1.25 M NaCl.
  • the washing buffer used also consists of PEG 8000 at 10% and NaCl but at 2.5 M.
  • the PROMEGA MagaZorb DNA mini prep kit is based on the protocol described in US Pat. No. 6,855,499.
  • the state of the art knows other commercial kits using magnetic beads in nucleic acid extraction and purification protocols.
  • the Silane Genomic DNA Kit from Life Technologies (Ref 370-12D) is proposed for the preparation of nucleic acids from blood samples.
  • alcohol of the ethanol or isopropanol type is used, which requires preparing the capture and wash pads by adding an alcohol before starting the extraction.
  • this protocol has other disadvantages that are incubation times greater than 3 minutes and 4 stages of DNA pellet-magnetic beads washing.
  • the inventors have realized that the commercial kits have considerably lower extraction and purification yields when they are used with other magnetic beads than those of the kit (see, for this purpose, example 5). of the experimental part below).
  • the inventors have set themselves the goal of proposing a simple process in terms of steps, preparatory protocols and reagents, for extracting and purifying nucleic acids, said method involving an adsorption or affinity chromatography step which can use any beads or particles, magnetic or paramagnetic.
  • the inventors have solved this technical problem and have found a solution to the disadvantages of the processes of the prior art by proposing a complete process making it possible to extract and purify nucleic acids from various samples by using lysis and polishing buffers. washing containing glycerol and this, using magnetizable particles of different origins.
  • the present invention provides a method for extracting and purifying at least one target nucleic acid contained in a sample, comprising the successive steps of:
  • step (b) contacting the cell lysate obtained after step (a) with magnetizable particles, under conditions allowing capture of said at least one target nucleic acid by said particles;
  • step (b) washing said magnetizable particles obtained following step (b) with a washing buffer preferably containing glycerol;
  • nucleic acid is meant, in the context of the present invention, a chromosome; a gene ; a regulatory polynucleotide; DNA, single-stranded or double-stranded, genomic, chromosomal, chloroplast, plasmidic, mitochondrial, recombinant or complementary; total RNA; messenger RNA; ribosomal RNA; a transfer RNA; an ANP; a portion or a fragment thereof.
  • nucleic acid used in the present invention is equivalent to the following terms and expressions: “nucleotide molecule”, “polynucleotide”, “nucleotide sequence” and “polynucleotide sequence”.
  • target nucleic acid (s) designates the nucleic acid (s) which it is desired to extract and purify by means of the claimed process.
  • sample used in the context of the process according to the present invention can be of very varied nature and source. In general, it is any sample for which it is desired to extract and purify the nucleic acids or it contains or by which it is contaminated.
  • this sample may be a biological fluid; a vegetable fluid such as sap, nectar and root exudate; a sample in a culture medium or in a biological culture reactor such as a cell culture of higher eukaryotes, yeasts, fungi, bacteria, viruses or algae; a liquid obtained from an animal or plant tissue; an animal or plant tissue; one or more cells; a cellular pellet; a sample taken from a food matrix, preferably diluted in a buffer; a sample taken from a chemical reactor; a sample from a treatment plant; a sample from a composting station; city, river, pond, lake, sea, swimming pool, air-cooled or underground water; a sample from a liquid industrial effluent; waste water, particularly from intensive livestock farms or chemical, pharmaceutical or cosmetic industries; a sample from an air filtration or coating; a sample on an object such as a piece of cloth, a coat, a sole, a shoe, a tool, a weapon, etc. ; a product pharmaceutical; a cosmetic product
  • sampling any type of sample collection, for example, by contact, scraping, drilling, cutting, punching, grinding, washing, rinsing, suction, pumping, etc ...
  • the biological fluid is preferably selected from the group consisting of blood such as whole blood or anti-coagulated whole blood, blood serum, blood plasma, lymph, saliva, sputum, tears, sweat, sperm, urine, stool, milk, cerebrospinal fluid, interstitial fluid, isolated bone marrow fluid, mucus or fluid of the respiratory tract, intestinal or genitourinary tract, cell extracts, extracts of tissues and organ extracts.
  • the biological fluid may be any fluid naturally secreted or excreted from a human or animal body or any fluid recovered from a human or animal body by any technique known to those skilled in the art such as extraction. , a sample or a wash. The steps of recovery and isolation of these different fluids from the human body or animal are performed prior to the implementation of the method according to the invention.
  • the sample used in the context of the present invention may contain, by its nature or possibly following contamination, one (or more) cells (identical or different).
  • cell is meant, in the context of the present invention, both a prokaryotic type of cell and eukaryotic type.
  • the cell may be a yeast such as a yeast of the genus Saccharomyces or Candida, a plant cell or an animal cell such as a mammalian or insect cell.
  • Prokaryotic cells are bacteria that can be Gram positive or Gram - positive.
  • bacteria belonging to the branches of spirochetes and chlamydiae mention may be made, by way of examples and in a non-exhaustive manner, of the bacteria belonging to the branches of spirochetes and chlamydiae, the bacteria belonging to the families of Enterobacteriaceae (such as Escherichia coli), Streptococcaceae (such as Streptococcus and, in particular, Streptococcus pneumoniae), microbacteria (such as Staphylococcus), legionellae, mycobacteria, bacillaceae and others.
  • Enterobacteriaceae such as Escherichia coli
  • Streptococcaceae such as Streptococcus and, in particular, Streptococcus pneumoniae
  • microbacteria such as Staphylococcus
  • legionellae mycobacteria
  • mycobacteria bacillaceae and others.
  • this implementation and in particular the extraction and purification method as defined below further comprises a preliminary step of preparing the sample with possible dissolution of the sample by the known techniques of those skilled in the art such as filtration, precipitation, dilution, distillation, mixing, concentration, etc.
  • Step (a) of the process according to the present invention consists, as in the known methods of extraction and purification of nucleic acids, in extracting the nucleic acids from the sample containing them while preserving their integrity, and in particular inhibiting or disabling enzymes that lyse target nucleic acids.
  • the lysis during step (a) is a chemical and enzymatic lysis step. Any lysis buffer for chemical and enzymatic lysis known to those skilled in the art can be used in the context of the present invention.
  • the lysis buffer implemented during step (a) of the process according to the invention contains glycerol.
  • the lysis buffer used in the context of the present invention is a buffer containing glycerol, at least one surfactant and at least one chaotropic agent.
  • the lysis buffer used in the context of the present invention advantageously contains glycerol in an amount of between 1 and 40%, in particular between 5 and 20%, in particular between 7 and 15% and, more particularly, order of 10% (ie 10% ⁇ 2%) by mass relative to the total mass of the lysis buffer (ie mass percentage). It is believed that the presence of glycerol in the lysis buffer is not necessary, but advantageous, as it increases the total yield of the protocol.
  • the lysis buffer used in the context of the present invention contains at least one surfactant, the latter participating in chemical lysis.
  • surfactant is understood to mean a molecule comprising a lipophilic (apolar) and a hydrophilic (polar) part. Any surfactant capable of solubilizing cell membranes and membrane lipids can be used in the context of the present invention.
  • this surfactant is chosen from anionic surfactants, cationic surfactants, zwitterionic surfactants, amphoteric surfactants and nonionic surfactants.
  • the lysis buffer according to the invention can comprise several surfactants belonging to the same family of surfactants previously listed (i.e. anionic, cationic, zwitterionic, amphoteric or nonionic) or several surfactants belonging to at least two distinct families of surfactants.
  • anionic surfactants are surfactants whose hydrophilic part is negatively charged such as alkyl or aryl sulfonates, sulfates, phosphates, sulfosuccinates or sarcosinates associated with a counterion such as an ammonium ion (NH 4 + ), a quaternary ammonium such as as tetrabutylammonium, and alkaline cations such as Na + , Li + and K + .
  • a counterion such as an ammonium ion (NH 4 + )
  • a quaternary ammonium such as as tetrabutylammonium
  • alkaline cations such as Na + , Li + and K + .
  • anionic surfactants it is possible, for example, to use tetraethylammonium paratoluenesulphonate, sodium dodecyl sulphate (or SDS), sodium laurylsarcosinate (or sarcosyl), sodium palmitate, sodium stearate, sodium myristate, sodium di (2-ethylhexyl) sulfosuccinate, methylbenzene sulfonate and ethylbenzene sulfonate.
  • the cationic surfactants are surfactants whose hydrophilic part is positively charged, in particular chosen from quaternary ammoniums comprising at least one C 4 -C 22 aliphatic chain associated with an anionic counterion chosen in particular from boron derivatives such as tetrafluoroborate or halide ions such as F “ , Br “ , “ or Cl “ .
  • Zwitterionic surfactants are neutral compounds having formal electric charges of one unit and opposite sign, in particular chosen from compounds having a C 5 -C 20 alkyl chain generally substituted with a negatively charged function such as a sulfate or a carboxylate and a function positively charged like an ammonium.
  • Zwitterionic surfactants include sodium N, N dimethyl dodecyl ammonium butanate, sodium dimethyl dodecyl ammonium propanate and amino acids.
  • Amphoteric surfactants are compounds that act as either an acid or a base depending on the medium in which they are placed. As amphoteric surfactants, it is possible to use disodium lauroamphodiacetate and betaines such as alkylamidopropylbetaine or laurylhydroxysulfobetaine.
  • Nonionic surfactants also known as neutral surfactants, are surfactants which have no group capable of being ionized in water at a neutral pH or near neutral. Such surfactants are, however, amphipathic since they contain lipophilic entities and hydrophilic entities.
  • the surfactant properties of nonionic surfactants, in particular hydrophilicity, are provided by uncharged functional groups such as an alcohol, an ether, an ester or an amide, containing heteroatoms such as a nitrogen atom or a nitrogen atom. oxygen atom. Because of the low hydrophilic contribution of these functions, the nonionic surfactant compounds are most often polyfunctional. Any nonionic surfactant known to those skilled in the art can be used in the context of the present invention.
  • alkoxylates of alkyls, fatty alcohols, fatty amines, fatty acids, oxoalcohols or alkylphenols can be used in the context of the present invention; ethoxylates of alkyls, fatty alcohols, fatty amines, fatty acids, oxoalcohols or alkylphenols; monoesters (monolaurate, monomyristate, monostearate, monopalmitate, monooleate, etc.), phospholipids and polyesters of fatty acids and glycerol; polyglycerolated fatty amides having an average of 1 to 5; oxyethylenated sorbitan fatty acid esters having from 2 to 30 moles of ethylene oxide; monoesters (monolaurate, monomyristate, monostearate, monopalmitate, monooleate, etc.) and polyesters of fatty acids and sorbitan, monoesters of polyoxyethylene
  • the (or) surfactant (s) used during step (a) are advantageously chosen from nonionic surfactants and anionic surfactants and mixtures thereof.
  • nonionic surfactants used in step (a) are advantageously chosen from polyethoxylated ethers, polyoxyethylene sorbitan monoesters and mixtures thereof.
  • the surfactant (s) used during step (a) are chosen from the group consisting of polyethylene glycol tert-octylphenyl ether (or Triton X- 100® ), polyoxyethylene monolaurate. sorbitan 20 (or Tween 20® ), sodium dodecyl sulphate (or SDS), sodium lauryl sarcosinate (or sarcosyl) and mixtures thereof.
  • the lysis buffer implemented during step (a) of the process according to the invention comprises a mixture of SDS and Tween 20® and advantageously a mixture of 1% SDS. 20% by mass and Tween 20 ® relative to the total mass of the lysis buffer.
  • the lysis buffer used in the context of the present invention contains at least one chaotropic agent, the latter also participating in chemical lysis.
  • chaotropic agent is meant, in the context of the present invention, a salt which modifies the solubility of proteins and may cause their precipitation.
  • Some of the chaotropic agents usable in the lysis buffer of the invention may further affect the three-dimensional structure of the proteins and denature them. Any chaotropic agent known to those skilled in the art can be used in the context of the present invention.
  • the chaotropic agent used in the context of the present invention is selected from the group consisting of sodium chloride (NaCl), ammonium sulfate ((NH 4 ) 2 SO 4 ), sodium perchlorate (NaClO 4 ), sodium iodide (NaI), lithium chloride (LiCl), lithium perchlorate (LiClO 4 ), barium (BaCl 2 ), cesium chloride (CsCl 2 ), potassium chloride (KCl), potassium iodide (Kl), urea (CO (NH 2 ) 2 ), a guanidine salt such as Guanidine thiocyanate (GuSCN) and mixtures thereof.
  • NaCl sodium chloride
  • Ammonium sulfate (NH 4 ) 2 SO 4 )
  • NaClO 4 sodium perchlorate
  • NaI sodium iodide
  • LiCl lithium chloride
  • LiClO 4 lithium perchlorate
  • the chaotropic agent used in the context of the present invention is a mixture of sodium chloride and guanidine thiocyanate.
  • the sodium chloride is at a concentration greater than or equal to 0.5 M and in particular of the order of 1 M (ie 1 M ⁇ 0.2 M) and the thiocyanate guanidine is at a concentration of between 0.5 and 1 M and in particular of the order of 0.85 M (ie 0.85 M ⁇ 0.1 M).
  • the lysis buffer implemented in the context of step (a) of the process according to the present invention may further comprise at least one element capable of inhibiting DNAses and RNAses and / or at least one element capable of destroying bridges. disulfide in proteins or to prevent their reformation.
  • said lysis buffer may further comprise at least one member selected from a thiol; a reducing agent such as ⁇ -mercaptoethanol or dithiothreitol (DTT); ethylene diamine tetraacetic acid (EDTA) and a citrate.
  • nucleic acids in the sample are not target nucleic acids and, in fact, must be removed, such as, for example, DNA or RNA
  • these compounds mention may be made of sodium hydroxide (NaOH), a RNAse and a DNAse.
  • the amount of lysis buffer used is such that the ratio (v / v) sample / lysis buffer is between 1/3 and 3, in particular between 1 / 2 and 2 and, in particular, this ratio is equal to 1.
  • step (a) of the process according to the present invention is a lysis not only chemical but also enzymatic, it is necessary to add to the mixture constituted by the sample and the lysis buffer, a protease and advantageously at least one endoprotease and this, to achieve enzymatic digestion (or enzymatic proteolysis) of the proteins of the sample and lead to the formation of fragments more or less short peptides from the proteins contained in the sample.
  • a protease and advantageously at least one endoprotease and this, to achieve enzymatic digestion (or enzymatic proteolysis) of the proteins of the sample and lead to the formation of fragments more or less short peptides from the proteins contained in the sample.
  • endoproteases specific or not, usable during step (a) of the method according to the invention.
  • the protease (s) used during step (a) of the process is (are) chosen from the group consisting of proteinase K, subtilisin, pepsin, trypsin, chymotrypsin, thrombin, thermolysin, HIV protease, elastase, factor Xa, a thiol endopeptidase such as capain or a caspase, a lysine-specific endopeptidase (endoLysN), a specific endopeptidase arginine (endoArgC) and endopeptidase specific for glutamic acid (endoGluC).
  • proteinase K subtilisin
  • pepsin trypsin
  • chymotrypsin chymotrypsin
  • thrombin thermolysin
  • HIV protease elastase
  • factor Xa a thiol endopeptidase
  • the protease more particularly implemented during step (a) of the process is a non-specific endoprotease and advantageously proteinase K. More particularly, this proteinase K is used at a concentration of between 0.1 and 5 mg / ml. , in particular between 0.4 and 2 mg / ml and in particular between 0.7 and 1.5 mg / ml of solution implemented in step (a).
  • solution implemented during step (a) is meant the solution formed from the sample, the lysis buffer and proteinase K.
  • the solution implemented during step (a) of the process according to the invention is homogenized manually or by vortexing and maintained at a temperature of between 40 and 65 ° C., in particular between 50 and 60 ° C. and, in particular, of the order of 56 ° C (ie 56 ° C ⁇ 3 ° C) for a period of between 2 and 30 min, in particular between 5 and 20 min and, in particular, of the order of 10 min (ie 10 min ⁇ 3 min).
  • a cell lysate containing the target nucleic acid (s) is obtained following step (a) of the process according to the present invention.
  • Step (b) consists in bringing the cell lysate obtained in step (a) into contact with magnetizable particles capable of reversibly capturing, by adsorption or via specific interactions, nucleic acids.
  • magnetizable particles is meant particles having magnetic properties only when subjected to a magnetic field. All magnetizable particles known to those skilled in the art can be used in the context of the present invention.
  • magnetizable particles used in the context of the present invention are substantially spherical and their average diameter is advantageously between 0.5 and 100 ⁇ and in particular between 1 and 50 ⁇ .
  • the terms "magnetizable particle”, “particle (para) magnetic”, “magnetizable ball” and “ball (para) magnetic” are equivalent and can be used interchangeably.
  • the magnetizable particles used in the context of the present invention comprise magnetic grains (or nanoparticles) and an organic or inorganic matrix.
  • magnetic grains or nanoparticles
  • organic or inorganic matrix Depending on the process used to prepare these magnetizable nanoparticles, it is possible to obtain different structures such as (i ') nanoparticles of heart / shell type with the core comprising the magnetic grains and the shell formed by the organic or mineral matrix or ( ⁇ ') nanoparticles in which the magnetic grains are uniformly distributed in the organic or mineral matrix.
  • the magnetic grains are in at least one material selected from the group consisting of metal oxides of iron, titanium, cobalt, zinc, copper, manganese or nickel; magnetite; Maghampte; hematite; ferrites of manganese, nickel or manganese-zinc and cobalt-nickel alloys.
  • the organic matrix of the magnetizable particles used in the context of the present invention is a natural or synthetic organic polymer and, for example, selected from the group consisting of albumin; cellulose and its derivatives in particular as defined in US Pat. No. 6,855,499; polyacrylates such as poly (acrylic acid) and poly (hydroxyethyl methacrylate); polyvinyl alcohol and polystyrene.
  • a mineral matrix is advantageously present in the form of silica gel.
  • the magnetizable particles used in the context of the present invention can capture the nucleic acids at their surface by adsorption.
  • this capture involves Van der Waals interactions, electrostatic interactions, hydrogen bonds or hydrophobic interactions.
  • the adsorption of the nucleic acids on the magnetizable particles is done by means of electrostatic interactions and hydrogen bonds.
  • the magnetizable particles have, at their surface, groups participating in such interactions or bonds, such as cationic or anionic groups. These groups are carried, intrinsically or by functionalization, by the particle matrix.
  • the magnetizable particles implemented in the context of the present invention can capture the nucleic acids to the surface through specific interactions.
  • This form of implementation requires the functionalization, covalent or otherwise, of the surface of the particles by an element capable of capturing the target nucleic acids such as, for example, by hybridization.
  • an element is advantageously chosen from the group consisting of an oligonucleotide complementary to a specific sequence present in the target nucleic acids, a poly (thymine) oligonucleotide, streptavidin, biotin or an anti-DNA antibody.
  • magnetizable particles possibly functionalized as previously defined.
  • commercial magnetizable particles may be used.
  • such particles include the Silanol balls (CHEMICELL), the balls MagPrep HS ® (Merck Estapor® SAS), Dynabeads ® MyOne TM beads (Life Technologies), the Dynabeads ® Streptavidin (Life Technologies) and MagaZorb ® beads (PROMEGA).
  • the ratio between the volume of sample during step (a) and the volume of magnetizable particles implemented during step (b) is between 1 and 50, in particular between 5 and 25 and, in particular, of the order of 10 (ie 10 ⁇ 2).
  • a capture buffer comprises at least one solvent selected from the group consisting of 1,2-butanediol; 1,2-propanediol; 1,3-butanediol; 1-methoxy-2-propanol acetate; 3-methyl-1,3,5-pentanetriol; a dibasic ester such as DBE-2, DBE-3, DBE-4, DBE-5 or DBE-6; diethylene glycol monoethyl ether (DGME); diethylene glycol monoethyl ether acetate (DGMEA); ethyl lactate; ethylene glycol; poly (2-ethyl-2-oxazoline); a sodium salt of a copolymer of 4-styrene sulfonic acid and maleic acid; te
  • the capture buffer implemented during said step (b) of the process according to the invention comprises at least two solvents as previously defined.
  • the capture buffer implemented during said step (b) of the process according to the invention is a mixture of two solvents as previously defined.
  • the capture buffer implemented during said step (b) of the process according to the invention is a mixture of TEG and 1,2-butanediol and, typically, a 60% (v / v) mixture of TEG and 40% (v / v) 1,2-butanediol.
  • the mixture used during step (b) of the process according to the invention containing the cell lysate, the magnetizable particles and the capture buffer as previously defined is homogenized manually or by vortexing and maintained at a temperature of between 10 ° C. and 40 ° C, advantageously between 15 and 30 ° C and, more particularly, at room temperature (ie 23 ° C ⁇ 5 ° C) and for a period of between 2 and 30 ° min, especially between 5 and 20 min and, in particular, of the order of 10 min (ie 10 min ⁇ 3 min).
  • the magnetizable particles have captured the nucleic acid (s) target (s).
  • Step (c) of the process according to the present invention consists in separating the magnetizable particles which have captured the target nucleic acids from the medium containing said particles at the end of step (b) and, in particular, of the other elements not captured by the magnetizable particles (ie not bound to these) present in this medium.
  • the latter is formed from the sample, the lysis buffer, the proteinase K and the capture buffer.
  • step (c) of the process according to the present invention consists in (ci) subjecting the magnetizable particles which have captured the target nucleic acid (s) to a magnetic field, (c 2 ) removing the liquid phase ie the medium surrounding said magnetizable particles, (c 3 ) adding a washing buffer and (c 4 ) resuspending said magnetizable particles in said wash buffer by suppressing the magnetic field, said steps (c 3 ) and (c 4 ) can be performed one after the other or simultaneously.
  • Steps (ci) to (c 4 ) are conventional steps in the various fields using magnetizable particles.
  • the magnetic field (or magnetic force) can be applied (e) during step (ci), by means of a magnet or a magnetic bench. Any technique for removing a liquid can be used in the context of step (c 2 ). By way of examples, this elimination can be carried out by inversion, tapping, absorption or aspiration.
  • Step (c) of the process according to the present invention and steps (ci) to (c 4 ) can be repeated at least once.
  • the washing buffer implemented during step (c) or (c 3 ) may be identical to or different from the washing buffer implemented during step (c) or (c 3 ) previous.
  • any washing buffer implemented in any of steps (c) or (c 3 ) contains glycerol, a surfactant and optionally a chaotropic agent as defined above.
  • the washing buffer (s) used in the context of the present invention contains (nen) glycerol in an amount of between 1 and 40%, in particular between 5 and 30% and, in particular, between 7 and 25% by weight relative to the total mass of the washing buffer.
  • the washing buffer (s) used in the context of the present invention contains (a) a surfactant, in particular a surfactant of the family of ethylenes glycols, for example a tetraethylene glycol (TEG) or a polyalkylene glycol.
  • a surfactant in particular a surfactant of the family of ethylenes glycols, for example a tetraethylene glycol (TEG) or a polyalkylene glycol.
  • TEG tetraethylene glycol
  • PEG polyethylene glycol
  • the polyethylene glycol in the washing buffer (s) has a molar mass greater than 1000 g / mol, in particular greater than 2000 g / mol, in particular between 4000 and 10000 g / mol and, more particularly, between 6000 and 8000 g / mol.
  • the washing buffer (s) used in the context of the present invention contains (a) a surfactant of the family of ethylene glycols and in particular tetraethylene glycol or polyethylene glycol in an amount of between 1 and 40%, especially between 5 and 30% and, in particular, between 7 and 25% by weight relative to the total mass of the washing buffer.
  • the solvent of the washing buffer (s) is typically selected from Tris at a concentration of between 5 and 50 mM, Tris base at a concentration between 5 and 50 mM, Tris / HCl at a concentration of between 5 and 50 mM, triethanolamine at a concentration between 5 and 50 mM or a mixture thereof.
  • the pH of the wash buffer is preferably of the order of 8 (i.e. 8 ⁇ 0.5).
  • the ratio between the volume of washing buffer implemented during each step (c) and the volume of magnetizable particles implemented during step (b) is between 10 and 200, in particular between 25 and 100, and in particular, of the order of 50 (ie 50 ⁇ 5).
  • Step (c) of the process according to the present invention and in particular steps (ci) and (c 4 ) are carried out at a temperature between 10 and 40 ° C, advantageously between 15 and 30 ° C and, more particularly, at room temperature (ie 23 ° C ⁇ 5 ° C).
  • the washing step is repeated twice.
  • the washing buffer used during the 1st washing step comprises glycerol, a surfactant such as a surfactant of the family of ethylenes glycols and a chaotropic agent as defined above and in particular glycerol, PEG and a chaotropic agent or glycerol , TEG and a chaotropic agent as previously defined, while the washing buffer used in the 2 nd washing step comprises glycerol and a surfactant as a surfactant in the family ethylene glycols as previously defined and in particular glycerol and PEG or glycerol and TEG as previously defined.
  • a surfactant such as a surfactant of the family of ethylenes glycols and a chaotropic agent as defined above and in particular glycerol, PEG and a chaotropic agent or glycerol , TEG and a chaotropic agent as previously defined
  • the washing buffer used in the 2 nd washing step comprises glycerol and a
  • Step (d) of the process according to the present invention consists in putting the target nucleic acid (s) back in solution by unclipping, by means of an elution buffer, the (or) the nucleic acid (s) target (s) of the magnetizable particles. During this stall, the non-covalent and low stability bonds between the target nucleic acid (s) and the magnetizable particles are broken by the action of the elution buffer, optionally combined with operating conditions of step (d) such as heating.
  • step (d) of the process according to the present invention consists in (di) subjecting the magnetizable particles to a magnetic field, (d 2 ) removing the washing buffer, (d 3 ) adding an elution buffer and ( d 4 ) resuspending said magnetizable particles in said elution buffer by suppressing the magnetic field, said steps (d 3 ) and (d 4 ) being performed one after the other or simultaneously.
  • Steps (di) to (d 4 ) are also conventional steps in the different fields using magnetizable particles.
  • the magnetic field (or magnetic force) can be applied during step (di) by means of a magnet or a magnetic bench. Any technique for removing a liquid can be used in the context of step (d 2 ). By way of examples, this elimination can be carried out by inversion, tapping, absorption or aspiration.
  • the elution buffer implemented during step (d) or (d 3 ) may be any elution buffer known to those skilled in the art and adapted to the type of capture implemented during the step ( b) prior to adsorption or specific interactions.
  • a buffer selected from Tris at a concentration of between 5 and 50 mM, Tris base at a concentration of between 5 and 50 mM, Tris / HCl. at a concentration of between 5 and 50 mM, triethanolamine at a concentration of between 5 and 50 mM or a mixture thereof.
  • the pH of the elution buffer is advantageously of the order of 9 (ie 9 ⁇ 0.5).
  • the ratio between the volume of elution buffer implemented during each step (d) and the volume of magnetizable particles implemented during step (b) is between 1 and 20, in particular between 2 and 10, and in particular, of the order of 5 (ie 5 ⁇ 1).
  • Step (d) of the process according to the present invention and in particular step (d 4 ) can be carried out at a temperature of between 40 and 80 ° C., in particular between 50 and 70 ° C. and, in particular, with 60 ° C (ie 60 ° C ⁇ 5 ° C), the steps (di) to (d 3 ) being, in turn, be carried out at a temperature between 10 and 40 ° C, preferably between 15 and 30 ° C and, more particularly, at room temperature (ie 23 ° C ⁇ 5 ° C).
  • step (d) of the process according to the present invention and in particular step (d 4 ) can be carried out with stirring and for a period of between 1 and 15 min, especially between 2 and 7 min and, in particular, of the order of 3 min (ie 3 min ⁇ 1 min).
  • This stirring is carried out by means of a magnetic stirrer and at a speed greater than or equal to 100 rpm, especially greater than or equal to 500 rpm and, in particular, between 1000 and 1500 rpm.
  • step (d) of the process according to the invention it is easy to recover the elution buffer containing the target nucleic acid (s) by eliminating the magnetizable particles and this, by applying a magnetic field.
  • the present invention also relates to the lysis and washing buffers that can be used in a process as defined above.
  • the present invention relates to a lysis buffer capable of being implemented in a process as defined above, containing at least one surfactant, at least one chaotropic agent and glycerol as previously defined.
  • a lysis buffer capable of being implemented in a process as defined above, containing at least one surfactant, at least one chaotropic agent and glycerol as previously defined.
  • a particular example of such a lysis buffer is given in the experimental section hereinafter.
  • the present invention relates to a washing buffer that can be used in a process as defined above, containing glycerol, a surfactant, especially a surfactant of the family of ethylene glycols such as a TEG or a polyalkylene glycol and optionally a chaotropic agent as defined above and in particular containing glycerol, TEG or PEG and optionally a chaotropic agent as defined above.
  • a washing buffer that can be used in a process as defined above, containing glycerol, a surfactant, especially a surfactant of the family of ethylene glycols such as a TEG or a polyalkylene glycol and optionally a chaotropic agent as defined above and in particular containing glycerol, TEG or PEG and optionally a chaotropic agent as defined above.
  • the present invention also relates to a kit comprising at least one lysis buffer as defined above and at least one washing buffer as previously defined.
  • the kit according to the invention comprises a lysis buffer and two wash buffers as given in the experimental part below or a lysis buffer and the washing buffer No. 2 such as given in the experimental part below.
  • the kit according to the present invention may further contain at least one member selected from the group consisting of (i ") a protease, especially an endoprotease and, in particular, proteinase K; (ii") a capture buffer such as previously defined; (iii ") an elution buffer as previously defined and (iv”) magnetizable particles as defined above.
  • the present invention further relates to the use of a lysis buffer, a wash buffer and a kit as previously defined for extracting and purifying target nucleic acids in a sample, said extraction and purification using particles. magnetizable.
  • the present invention relates, on the one hand, to a method for lysing a sample comprising contacting said sample with a lysis buffer according to the invention and, on the other hand, a method for washing the samples. particles magnetizable particles having captured at least one target nucleic acid comprising contacting said particles with a washing buffer according to the invention.
  • Figure 1 is a schematic representation of the method according to the present invention.
  • Figure 2 shows the percent purification of the DNA as a function of the amount of glycerol contained in the lysis buffer and the wash buffers.
  • Figure 3 shows the percentage of purification of the DNA according to the amount of glycerol contained in the lysis buffer.
  • Figure 4 shows the percentage of purification of the DNA as a function of the amount of glycerol contained in Wash Buffer No. 1.
  • Figure 5 shows the percentage of purification of the DNA as a function of the amount of glycerol contained in Wash Buffer No. 2.
  • FIG. 6 shows the threshold cycle or Ct value for a supernatant obtained with the PROMEGA commercial process using the magnetic beads referred to as the MagaZorb beads or other non-referenced commercial beads that are the MyOne Dynabeads beads.
  • Figure 7 shows the threshold cycle or Ct value for a supernatant obtained with the method according to the present invention using different magnetic beads.
  • wash buffer (2 M GuSCN, 10 mM Tris-HCl pH 8, PEG8000 10%, Glycerol A%);
  • the DNA is analyzed by qPCR using primers specific for the targeted pathogens.
  • DNA extraction is obtained as soon as 1% of glycerol is added, for certain bacterial samples. It is the same for the protocol using 20% glycerol in the lysis and washing buffers. The best extraction results for all samples are obtained when the lysis buffer and wash buffers contain 10% glycerol.
  • Example 2 Variation of the concentration of glycerol present in the lysis buffer.
  • the concentration in the lysis buffer denoted "B%" and corresponding to the glycerol mass with respect to the total mass of solution represents 0%, 1%, 10% or 20%.
  • wash buffer (10 mM Tris-HCl pH 8, PEG8000 20%, Glycerol 20%);
  • the DNA is analyzed by qPCR using primers specific for the targeted pathogens.
  • wash buffer (2M GuSCN, 10mM Tris-HCl pH 8, PEG8000 10%, Glycerol C%);
  • wash buffer (10 mM Tris-HCl pH 8, PEG8000 20%, Glycerol 20%);
  • the DNA is analyzed by qPCR using primers specific for the targeted pathogens.
  • the DNA is analyzed by qPCR using primers specific for the targeted pathogens.
  • Example 5 PROMEGA commercial extraction protocol set with different beads.
  • the quantity of amplicons produced at each cycle is monitored by measuring the fluorescence.
  • the threshold cycle or Ct for "cycle treshold"
  • the results of the Ct supernatants obtained by the PROM EGA protocol using the magnetic beads referenced (MagaZorb, PROM EGA) or unreferenced commercial beads (Dynabeads MyOne, Life Technologies) are given in FIG. 6.
  • the eluate obtained contains the DNA of three pathogens (E. coli, S. epidermidis, S. pneumoniae).
  • Figure 6 shows the Ct obtained for each pathogen.
  • Example 6 Process according to the invention with magnetic beads from different suppliers.
  • wash buffer (2M GuSCN, 10mM Tris-HCl pH 8, PEG8000 10%, Glycerol 10%);
  • wash buffer (10 mM Tris-HCl pH 8, PEG8000 20%, Glycerol 20%);
  • Figure 7 shows the Ct obtained, corresponding to the DNA extracted from E.

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WO2017041005A1 (en) * 2015-09-03 2017-03-09 Abbott Molecular Inc. Hybridization buffers comprising an alkyl diester
US10457981B2 (en) 2016-01-08 2019-10-29 Abbott Molecular Inc. Hybridization buffers
US20180135040A1 (en) 2016-02-16 2018-05-17 Life Magnetics, Inc. Methods for separating nucleic acids with graphene coated magnetic beads
GB201617388D0 (en) 2016-10-13 2016-11-30 Randox Laboratories Limited Method of extracting material from a fluid and extractor
FR3066113B1 (fr) * 2017-05-12 2020-06-05 ISP Investments LLC. Procede d’obtention d’un extrait aqueux d’anethum graveolens enrichi en petits arn
US20210163969A1 (en) * 2018-08-06 2021-06-03 Northwestern University Combined transcription and translation platform derived from plant plastids and methods for in vitro protein synthesis and prototyping of genetic expression in plants
JP6942223B1 (ja) * 2020-06-12 2021-09-29 ジェネシスヘルスケア株式会社 唾液サンプリングキット
CN115197935B (zh) * 2021-04-08 2025-10-21 上海细胞治疗集团股份有限公司 纤维素色谱纯化rna的方法
CN115109115B (zh) * 2021-04-12 2025-06-27 中国热带农业科学院热带生物技术研究所 一种通过硅胶柱吸附洗脱快速纯化蛋白的方法
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