WO2000077235A1 - Agent et procede permettant d'isoler es acides nucleiques extra-chromosomiques - Google Patents

Agent et procede permettant d'isoler es acides nucleiques extra-chromosomiques Download PDF

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Publication number
WO2000077235A1
WO2000077235A1 PCT/US2000/016712 US0016712W WO0077235A1 WO 2000077235 A1 WO2000077235 A1 WO 2000077235A1 US 0016712 W US0016712 W US 0016712W WO 0077235 A1 WO0077235 A1 WO 0077235A1
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sodium
nucleic acids
solution
dna
group
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PCT/US2000/016712
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English (en)
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Thomas D. Reed
John R. Dedman
Marcia A. Kaetzel
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University Of Cincinnati
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Priority to AU56206/00A priority Critical patent/AU5620600A/en
Publication of WO2000077235A1 publication Critical patent/WO2000077235A1/fr

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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

Definitions

  • the invention relates to compositions and methods for isolating extra-chromosomal nucleic acids from biological materials. More particularly, the invention relates to plasmid DNA isolation methods employing non-toxic chaotropic agents.
  • Extra-chromosomal DNA is distinct from chromosomal or genomic DNA that forms the chromosomes.
  • genomic DNA is attached or bound to a membrane.
  • eukaryotes such as mammalian or yeast cells
  • the genomic DNA is contained within and bound to the nuclear membrane.
  • prokaryotes such as bacteria
  • genomic DNA is bound to the plasma membrane surrounding the cytoplasm. While all cells contain genomic DNA, only some have extra-chromosomal DNA.
  • extra- chromosomal DNA is not attached to the chromosomal (genomic) DNA, and as such, is not attached to cell membranes.
  • Extra-chromosomal DNA may be naturally occurring or artificially introduced into eukaryotic or prokaryotic cells.
  • Extra- chromosomal DNA forms known and used in genetic cngincci ing 01 cloning include, but are not limited to, plasmid, cosmid, bacterial artificial chromosome (BAC), and yeast artificial chiomosoinc (YAC) DNA
  • cells or bacte ⁇ a are lysed to harvest genomic DNA by completely disrupting all cell membranes These membranes include the plasma membranes, which form the outer wall of the cell, and the intracellular membranes, including the nuclear membrane In this manner, genomic DNA is released from its membrane-bound state into the lysate
  • cells or bacte ⁇ a are treated with an alkaline lysis, which is a more gentle and incomplete disruption of the cell membranes
  • an alkaline lysis genomic DNA remains tethered to the membranes, while extra-chromosomal DNA is icleased into the lysate Sedimentation of the cellular deb ⁇ s into a pellet effectively removes genomic DNA from the lysate, since it remains bound to
  • Biological mate ⁇ als can include homogenized tissue samples, yeast or bacte ⁇ al cultures, tissue culture cells originating from complex organisms such as mammals or insects but subsequently giown //; vit/ o, and agarosc gel containing semi-pure DNA preparations
  • Chaotropic agents similar to the ones used in the present invention have been previously used only in the isolation of genomic DNA.
  • Homogenized tissue samples or cell suspensions are treated with chaotropic agents, such as those disclosed in the published application W097/05248, to lyse the cell membranes and release genomic DNA into the lysate. If such materials are used to harvest extra-chromosomal DNA, the resultant release of genomic DNA contaminates the extra-chromosomal DNA.
  • others have attempted to use such materials for isolating plasmid DNA, but have failed to devise the needed modifications to the materials and methods. Therefore, there are no prior art references teaching the use of chaotropic agents for plasmid or other extra-chromosomal DNA preparations.
  • a chaotropic solution has been modified specifically for the isolation of extra-chromosomal DNA.
  • the process of the invention does not include lysis of cell homogenates or bacterial suspensions with chaotropic agents.
  • the cells or bacteria are lysed using a standard alkaline lysis method.
  • the lysate is separated from the cellular debris and genomic DNA, and then treated with the chaotropic agents as a purification step.
  • the yield of extra-chromosomal DNA isolated using the present invention materials and method is significantly greater and purer than those using conventional methods.
  • the time of isolation using the present invention method is comparable to those dependent upon the use of DNA binding matrices.
  • the yield is also enhanced because the method of the present invention is not influenced by the electrostatic, temperature and topological variables inherent in DNA-binding resin isolation procedures. Since the present invention requires only aqueous solutions of relatively inexpensive chemicals, the cost is significantly less than other methods. Perhaps the greatest benefit of the present invention is that the extra-chromosomal DNA is isolated in an undegraded and intact condition, providing a more useful final product.
  • the present invention provides methods and materials for isolating substantially pure and undegraded extra-chromosomal nucleic acids from biological materials.
  • the solution contains effective amounts of a chaotropic agent, a buffer present in an amount sufficient to maintain an alkaline pH equal to or greater than about 7.5, salt, detergent, and alcohol.
  • the invention is also a method for isolating substantially pure and undegraded extra-chromosomal DNA from biological materials.
  • the steps in the method include precipitating extra-chromosomal DNA and contaminating RNA from a cell lysate, treating the extra-chromosomal DNA with RNase to digest the contaminating RNA, removing residual contaminants from extra- chromosomal DNA by adding the chaotropic solution, precipitating the DNA with an alcohol, pelleting the DNA by sedimentation, and washing the sedimented DNA with alcohol.
  • An alternative use of the invention is for recovery of any type of DNA from agarose gel preparations.
  • lysis is meant cellular dissociation involving physical disruption and breakage of the cell wall and/or membrane, causing intracellular components including nucleic acids to be released into the surrounding medium
  • incomplete lysis is meant the lysis of a cell or a group of cells which renders the cell chromosomes, cell membranes and other components capable of forming a precipitate while leaving the extracellular DNA in solution
  • alkaline lysis is meant the breaking open of a cell or a group of cells, or the liberation of some or all of the intracellular components following treatment of the cell or cells with an alkali or base which partially digests the organism's cell wall or membrane. This type of lysis breaks open a cell or a group of cells, and renders the cell chromosomes, cell membranes and other components capable of forming a precipitate while leaving the extracellular DNA in solution
  • enzyme lysis is meant the breaking open of a cell or a group of cells, or the liberation of some or all of the intracellular components following treatment of the cell or cells with an enzyme which partially digests the organism's cell wall This type of lysis breaks open a cell or a group of cells, and renders the cell chromosomes, cell membranes and other components capable of forming a precipitate while leaving the extracellular DNA in solution.
  • detergent or “surfactant” is meant a molecule or class of molecules which have a hydrophobic region or moiety capable of interacting with hydrophobic solvents and the hydrophobic portions of cellular membranes, and a hydrophihc region or moiety which may have a positive or a negative charge in solution, or alternately may have a polar region with no charge at all.
  • release of nucleic acids is intended to mean the liberation of nucleic acids in sufficient quantities such that the method of release is useful for further purification and subsequent use in molecular biology.
  • nucleic acid or “nucleic acids” is meant polydeoxyribonucleotides or polyribonucleotides of at least two, and preferably 10 or more nucleotides in length.
  • nucleic acid includes polynucleotides, oligonucleotides, and DNA or
  • RNA molecules can refer to either single-stranded or double- stranded polynucleotides, or both.
  • Biological sample any specimen or sample containing substances of biological or biochemical origin.
  • Biomaterials include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA or BACs; yeast transformed with yeast expression vectors or YACs; insect cell systems infected with viral expression vectors (e.g. baculovirus); plant cell systems transformed with viral expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), or cells derived from higher organisms such as mammalian tissue culture cells transfected with recombinant plasmid DNA or infected with viral expression vectors.
  • Biological material may also refer to agarose or polyacrilamide gel preparations used to separate any nucleic acids by size, charge or other characteristic.
  • substantially purified refers to recovery of nucleic acid which is at least 80% and preferably 90-95% purified with respect to removal of a contaminant, e.g., cellular components such as protein, lipid or salt; thus, the term “substantially pu ⁇ fied” generally refers to separation of a majority of cellular proteins or reaction contaminants from the sample, so that compounds capable of lntci lciing with the subsequent use of the isolated nucleic acid are removed
  • buffer refers to a buffeted solution that resists changes in pH by the action of its acid-base conjugate components
  • chaotropic environment refers to an environment which contains approp ⁇ ate chaotropic agents, such as urea in sufficient concentration to disrupt the tertiary structure of proteins, or which is maintained at a temperature or other condition which causes such disruption Chaotropic agents or conditions such as temperature and pH may disrupt structure in a va ⁇ ety of ways, including the disruption of hydrogen bonds
  • Suitable chaotropic environments include 2-8M urea, 4-7M guanidmium, detergents such as SDS at concentrations around 0 1% by weight, and acids such as acetic acid at concentrations of about 1M, basic conditions of, e g , pH 1 1 and above, and elevated temperatures
  • the invention encompasses solution and methods for isolating substantially pure and undegraded extra-chromosomal nucleic acids from biological materials.
  • the present invention is an entirely liquid based system that will not require the production or use of columns or beads employing DNA-binding materials. No toxic chemicals such as phenol or chloroform will be needed.
  • the use of the present invention to isolate plasmid or other extra-chromosomal DNA is significantly less expensive and as fast or faster than methods currently known and available.
  • the methods can be adapted to differing amounts of biological materials by changing volumes of the reagents used.
  • plasmid DNA can be harvested from bacterial cultures in what are conventionally known as "mini-", "midi-", or "maxi- prep" amounts.
  • the extra-chromosomal DNA isolated using the materials and methods of this invention is of particularly high purity, quality, and thus, usefulness.
  • the present invention is distinct from the prior art disclosures of chaotropic agents used to purify DNA because those materials and methods were designed for the isolation of genomic DNA. As such they use a formulation and method that will disrupt cell membranes and release both genomic and any extra-chromosomal DNA into a heterogeneous mixture. Isolation of extra-chromosomal DNA from such a mixture is, at best, an impractical and highly inefficient process.
  • the present invention is also distinct from those in the pnor art that are directed to the isolation of cxtiachiomosomal DNA
  • cells or bacte ⁇ a are first lysed using a standard alkaline lysis method Alkaline lysis releases cxtrachiomosomal-DNA and RNA fiom the cells or bacte ⁇ a into the crude lysate supernatant, but genomic DNA remains membrane-bound and is pelleted in the bacterial debris along with the membranes with centnfugation RNA is digested by adding RNase to the lysate
  • the RNasc-ticatcd lysate is applied to column or beads containing the DNA-binding materials, washed to remove protein contaminants, and eluted
  • the DNA in a lysate is separated from contaminants first by ultracentnfugation on a gradient, such as cesium salts, and then dialyzed and/or
  • a lysate is first prepared using a standard alkaline lysis method
  • the neutralized solution is treated with an alcohol precipitation followed by RNase to degrade contaminating RNA in the lysate
  • a chaotropic solution is added to the lysate to remove residual protein and degraded RNA contaminants from the extra-chromosomal DNA
  • a typical method of the present invention involves the recovery of pu ⁇ fied plasmid from a bacte ⁇ al lysate Accordingly, typical pu ⁇ fication procedures first require the preparation of the bacte ⁇ al lysate from a bacte ⁇ al culture Those skilled in the art are familiar with methods for lysing bacte ⁇ a The present invention is described in terms of the known alkaline lysis procedure, however, any procedure which effectively lysis the bacterial cells and releases plasmid DNA is applicable, such as enzymatic lysis. Typically these procedures involve adding a bacterial pellet to solutions which break open the bacterial cells, and cause the bacterial chromosomes, cell membranes and other components to form a precipitate while leaving the plasmid DNA in solution.
  • the alkaline lysis is generally prepared by pelleting a suspension of a biological material, such as a liquid culture of bacteria, through centrifugation. The supernatant is poured off and the pellet resuspcnded in buffer solution. The biological material is then lysed by addition of a strong base, e.g., sodium hydroxide, in the presence of a detergent such as sodium dodecyl sulfate (SDS). The alkaline lysis is terminated by the addition of a neutrilzation solution such as sodium or potassium acetate. The neutralized lysis mixture is centrifuged to pellet all cellular debris, including membranes and membrane-bound chromosomal DNA.
  • a biological material such as a liquid culture of bacteria
  • the lysate can alternatively be separated from the cellular debris by various filtration methods well known in the art, e.g., by gravity or by vacuum.
  • the supernatant (“lysate”) is poured into a clean tube for the precipitation step.
  • Alcohol such as ethanol
  • Alcohol is added to the lysate for a final concentration of about 70%.
  • the solution is centrifuged to pellet the nucleic acids, which will include RNA and extra-chromosomal DNA. The supernatant is discarded.
  • the pellet is resuspended in a buffer solution containing RNase to degrade all RNA contaminants.
  • the concentration of the buffer solution is generally an amount such that the pH is from about 7 to about 12, preferably where the pH is from about 7 to about 10, and most preferably where the pH is from about 7 to about 8.
  • the RNase incubation is generally performed at 37° (but can be performed at room temperature) for about 10 minutes. 3.
  • the RNase-treated solution is then mixed with a chaotropic solution and stored at about room temperature for about 10 minutes.
  • the mixture of the RNase-treated solution and the chaotropic solution is then treated with a solvent (0.3 to 1.0 of solution volume) in order to precipitate the extra-chromosomal DNA, and subsequently centrifuged to pellet the DNA away from contaminating proteins and degraded RNA.
  • the supernatant is removed and discarded.
  • the DNA pellet is washed with a solvent (about 60% to about 80%) to remove any residual traces of the chaotropic solution. Following centrifugation, the supernatant is removed and the DNA pellet is allowed to dry.
  • the DNA pellet is then dissolved in water or any suitable buffer.
  • the chaotropic solution is generally an aqueous mixture of effective amounts of one or more chaotropic agents.
  • Any chaotropic agent is useful in the practice of the present invention including but not limited to guanidine thiocyanate (GnSCN), sodium iodide, sodium perchlorate, guanidine hydrochloride, urea, hydroxides such as sodium or potassium hydroxide, guanidine salt, potassium thiocyanate, formamide, and sodium chloride.
  • Chaotropic agents include a combination of these reagents, such as a mixture of base with urea or guanidine hydrochloride.
  • Preferred embodiments of the present invention use aqueous solutions of 6 M guanidine hydrochloride. Best results are achieved when the chaotropic agent is present in relatively high concentrations ranging from 1 M to 12 M
  • Preferred chaotropic agents for the solution include guanidine thiocyante, guanidine hydrochlo ⁇ de, sodium iodide, and mixtures thereof
  • concentration of chaotiopes in the solution is preferably in the range of from about 1 M to about 7 M and is more preferably in the range of from about 2 M to about 5 M
  • the chaotropic solution generally comp ⁇ ses one or more chaotropic agents and optionally contains one or more compounds selected from the group consisting of buffers or buffer solutions, solvents, surfactants, salts, preservatives, anti-oxidants, bacte ⁇ ostats, solutes, reducing agents, stabilizers and mixtures thereof
  • Representative acceptable buffers comprise nontoxic buffer and solution thereof, solutions that resist change of pH thereby giving stability to the components present in the solution
  • the buffer and solutions thereof can be used alone and, in a presently preferred embodiment, the buffer and solution thereof is used in combination with acceptable aqueous miscible fluids
  • Typical buffer aqueous solutions comprise sodium dihydrogen phosphate and disodium monohydrogen phosphate, sodium dihydrogen phosphate , disodium monohydrogen phosphate and sodium chlo ⁇ de , sodium carbonate , sodium monohydrogen carbonate and sodium chlo ⁇ de , potassium dihydrogen phosphate and sodium monohydrogen phosphate, potassium dihydrogen
  • the buffer aqueous solution in another embodiment can comprise a sole component such as sodium phosphate monobasic, sodium phosphate dibasic, potassium hydrogen tartrate, potassium dihydrogen citrate, potassium hydrogen phthalate, sodium tetraborate, sodium carbonate, sodium hydrogen carbonate and mixtures thereof.
  • solvent refers to alcohols and polar aprotic solvents.
  • Alcohols are meant in the sense of the commonly used terminology for alcohol, preferably lower alcohols with 1 to 10 carbon atoms, more preferably methanol, ethanol, iso-propanol, n-propanol, or t-butanol, as well as glycerol, propylene glycol, ethylene glycol, polypropylene glycol, and polyethylene glycol, and most preferably ethanol or iso-propanol.
  • Such alcohols are solvents that, when added to aqueous solution, increase the hydrophobicity of the solution by decreasing solution polarity.
  • Polar aprotic solvents are such molecules as dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-methylpyrrolidone (NMP), tetrahydrofuran (THF), dioxane, acetonitrile, etc., that can be used in place of or in addition to the alcohol.
  • a lower alcohol such as ethanol, propanol or butanol, is also included, at from about 5% to about 40% by volume and more preferably at from about 15% to about 20% by volume.
  • Representative acceptable surfactants for the present purpose comprise anionic, cationic, amphoteric and nonionic surfactants.
  • surfactants comprise sorbitan trioleate, sorbitan tristearate, propylene glycol monostearate; sorbitan sesquiolate; glycerol monostearate; sorbitan monooleate; propylene glycol monolaurate; sorbitan monostearate; diethylene glycol monostearate, glycerol monostearate, die hylmonolaurate, sorbitan monopalmitate, sorbitan monolaurate, TRITONS, polyoxyethylene ethers, polyoxyethylene lauryl ether, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tnstearate, polyoxyethylene sorbitan t ⁇ oleate, polyoxyethylene glycol monooleate, polyoxyethylene glycol monostearate, t ⁇ ethanolamine oleate, polyoxyethylene monyl phenol, polyethylene glycol monolaurate, polyoxyethylene sorbitan triste
  • Acceptable salts include, but are not limited to, those prepared from the following acids hydrochlo ⁇ c, hydrobromic, sulfu ⁇ c, nitric, phosphonc, maleic, acetic, salicylic, p-toluene sulfonic, tarta ⁇ c, citric, methane sulfonic, formic, malonic, succmic, naphthalene-2-sulfon ⁇ c, and benzene sulfonic
  • acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium, lithium or calcium salts (potassium chlonde, sodium acetate, sodium nit ⁇ te, lithium chlo ⁇ de, and sodium bromide )
  • the salt concentration is generally m the range of about 0 01 M to about 1 M and preferably in the range of about 0 1 M to about 0 5 M
  • reducing agent refers to a compound that, in a suitable concentration in aqueous solution, maintains sulfhydryl groups so that the intra- or lntermolecular disulfide bonds are chemically disrupted
  • suitable reducing agents include dithiothreitol (DTT), dithioeryth ⁇ tol (DTE), beta- mercaptoethanol (BME), cysteine, cysteamine, thioglycolate, glutathione, and sodium borohyd ⁇ de While emphasis is placed on thiol-contai ng compounds, any material that is capable of the disulfide to thiol conversion without undesirable side reactions is included in this definition
  • At least one antioxidant is added to the solutions of the present invention
  • the antioxidants arc preferably BHA, BHT, octyl or dodecyl gallate, SO 2 , for instance in the form of sodium sulfites or sodium thiosulfite, lactic acid, cit ⁇ c acid, tarta ⁇ c acid and/or the salts thereof, vitamin C, vitamin E and u ⁇ c acid and the salts thereof
  • physiologically tolerated antioxidants are alpha -tocopherol, bi rubin, vitamin C, vitamin E, uric acid, and the salts and/or de ⁇ vatives thereof
  • Uric acid and the salts thereof are preferably added in a concentration of from about 0 01 to about 1 wt %, vitamin C in a concentration of 0 from about 0 01 to 2 wt %, and vitamin E in a concentration of from about 0 01 to about 0 1 wt %
  • the preservatives used may be formic acid in a concentration of preferably from about 0.
  • propiomc acid, lactic acid and sorbic acid in a concentration of preferably 0.05 to 6 wt. %, especially preferred is from about 0.05 to about 12 wt. %, SO 2 (preferably from about 0.01 to about 0.6 wt %), salicylic acid and the salts thereof (preferably from about 0 01 to about 0 5 wt. %), PBH ester (preferably from about 0.05 to about 0.6 wt. %), lmidazohdinyl urea derivatives (preferably from about
  • Stenhzation can be accomplished by any art-recognized technique, including but not limited to, filtration or addition of antibactenal or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • antibactenal or antifungal agents for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal.
  • isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • Extra-chromosomal DNA or fragments of genomic DNA can be partially530ificd by conventional agarose or acrylamide gel electrophoresis on the basis of size, charge, or other properties. Since these mate ⁇ als are unlikely to contain RNA contaminants, no RNase treatment steps are needed.
  • the region of the gel containing the DNA of interest is excised and solubhzed using an enzyme that specifically cleaves the gel matnux bonds without damaging DNA, e g , beta-agarose. After solubhzation, the gel and DNA solution can be treated with the chaotropic solution of the present invention to remove gel components and other contaminants.
  • the chaotropic solution is added to the DNA/gel solution at a ratio of about 10 1, and stored at about room temperature for about 10 minutes
  • An alcohol (from about 0 3 to about 1 0 volumes relative to volume of the mixture of chaotropic solution and the DNA/gel solution) is added to the mixture in order to promote precipitation of the DNA
  • This mixture is cent ⁇ fuged to pellet the DNA
  • the supernatant is removed and discarded
  • the DNA pellet is washed with ethanol or 2-propanol to remove any residual traces of the chaotropic solution
  • the effective final concentration of alcohol is from about 60% to about 80%. Following cent ⁇ fugation, the supernatant is removed and the DNA pellet is allowed to dry The DNA pellet is then dissolved in water or any suitable buffer
  • the present invention also contemplates a icagcnl system, typically in kit fo ⁇ n, that can be utilized in carrying out the before-described isolation methods
  • the system includes, in an amount sufficient for at least one isolation, a separately packaged reagent for a process for the isolation of extra-chromosomal nucleic acids in a biological sample Instructions for use of the packaged icagent arc also typically included
  • the term "package” refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits reagents of the present invention
  • "Instructions for use” typically include a tangible expression desc ⁇ bmg the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time pe ⁇ ods for reagent/sample admixtures, temperature, buffer conditions and the like
  • the packaging matenals discussed herein in relation to diagnostic systems are those customanly utilized in diagnostic systems Such materials include glass and plastic (e g , polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like
  • the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which compnses (a) a first container compnsmg one of the chaotropic agents of the present invention, and (b) one or more other containers compnsmg one or more of the following wash reagents, lysis reagents, Rnase, Rnase digestion buffer, and polyacrylamide or agarose-gel solubihzation solution
  • a compartmentalized kit includes any kit m which reagents are contained in separate containers Such containers include small glass containers, plastic containers or st ⁇ ps of plastic or paper Such containers allows one to efficiently transfer reagents from one compaitmcnl lo anothci compartment such thai the samples and reagents are not cross-contaminalcd, and the agents or solutions of each conlainci can be added in a quantitative lashion iiom one compaitmcnl to another
  • Such containers will include a container which will accept the test
  • Suitable culture media include ste ⁇ le preparations of LB or 2XYT broth, which are widely known and used in the propagation of bacte ⁇ a.
  • 100 ml of broth is placed in a sterile 500 ml culture flask
  • the broth is innoculated with E coli harbo ⁇ ng the plasmid of interest, and the flask is placed m a shaking incubator overnight at 37°
  • the culture is transferred to a cent ⁇ fuge tube and the bactena are pelleted by centifuging for 5 minutes at about 1900 g. This can be done in a SORVALTM RC5B model centrifuge, using an
  • the precipitated pellet obtained from the lysate is rcsuspendcd in about 1 -2 ml of 10 mM Tris-HCl pH 7.5 buffer for the RNase treatment step.
  • Suitable culture media include sterile preparations of LB or 2XYT broth, which are widely known and used in the propagation of bacteria. Typically, 30 ml of broth is placed in a sterile 150 ml culture flask. The broth is inoculated with E. coli harboring the plasmid of interest, and the flask is placed in a shaking incubator overnight at 37°. The following day, the culture is transferred to a 50 ml FALCON® tube and the bacteria are pelleted by centifuging for 5 minutes at 1500 g. This can be done in a Beckman centrifuge, using an JA20 rotor at 2000 rpm.
  • Example 5 Prepare a 2 ml culture of E. coli harboring the plasmid DNA of interest.
  • Suitable culture media include sterile preparations of LB or 2XYT broth, which are widely known and used in the propagation of bacteria.
  • 2 ml of broth is placed in a sterile 5 ml culture tube.
  • the broth is innoculated with E. coli harboring the plasmid of interest, and the flask is placed in a shaking incubator overnight at 37°. 0
  • the bacteria are pelleted by centifuging for 2 minutes at maximum speed (12000-15000 rpm) in a microcentrifuge at room temperature.
  • microcentrifuge tube in a microcentrifuge at maximum speed (typically 12,000-15,000 rpm) for 5-10 minutes at 4°.
  • Fragments of chromosomal DNA are partially purified by conventional agarose gel electrophoresis on the basis of size.
  • the region of the gel containing the DNA of interest is excised and placed in a microcentrifuge tube containing 3.0:0.2 Tris EDTA buffer. 3. The tube is placed in a 55° heat block until the gel is melted for about 10 minutes.
  • Precipitate the DNA by adding 500 ⁇ l ice-cold 95% ethanol and storing on ice for 10 minutes. Centrifuge the mixture of the chaotropic solution and the DNA/gel solution is to pellet the DNA. This can be done by placing the microcentrifuge tube in a microcentrifuge at maximum speed (typically 12,000-15,000 rpm) for 5- 10 minutes at 4°.
  • the DNA pellet is then dissolved in water or any suitable buffer.

Abstract

Cette invention concerne des matériaux et des procédés permettant d'isoler sélectivement des acides nucléiques extra-chromosomiques à partir de matériaux biologiques. L'obtention de ces acides nucléiques extra-chromosomiques ainsi isolés se fait sans recours à des substances chimiques toxiques ou à des matrices liant l'ADN, en faisant intervenir un agent chaotrope au stade de purification finale. Cette invention convient particulièrement bien pour l'isolation rapide d'ADN plasmidique pur et intact à partir de cultures bactériennes.
PCT/US2000/016712 1999-06-16 2000-06-16 Agent et procede permettant d'isoler es acides nucleiques extra-chromosomiques WO2000077235A1 (fr)

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US60/139,423 1999-06-16

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US7608642B2 (en) 2002-12-16 2009-10-27 Kimberly-Clark Worldwide, Inc. Wound and skin care compositions
WO2009144182A1 (fr) * 2008-05-30 2009-12-03 Qiagen Gmbh Réactif de lyse, de liaison et/ou de lavage, utilisable pour isoler et/ou purifier des acides nucléiques
CN101124321B (zh) * 2004-11-05 2012-02-29 恰根北美控股有限公司 用于从稳定剂中纯化核酸的组合物和方法
US8586306B2 (en) 2009-02-18 2013-11-19 Streck, Inc. Preservation of cell-free RNA in blood samples
US9956281B2 (en) 2011-05-04 2018-05-01 Streck, Inc. Inactivated virus compositions and methods of preparing such compositions
CN108531472A (zh) * 2017-03-05 2018-09-14 北京天健惠康生物科技有限公司 一种用于核酸提取的裂解液
US10091984B2 (en) 2013-07-24 2018-10-09 Streck, Inc. Compositions and methods for stabilizing circulating tumor cells
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