WO2001034844A1 - Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode - Google Patents

Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode Download PDF

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WO2001034844A1
WO2001034844A1 PCT/US2000/031005 US0031005W WO0134844A1 WO 2001034844 A1 WO2001034844 A1 WO 2001034844A1 US 0031005 W US0031005 W US 0031005W WO 0134844 A1 WO0134844 A1 WO 0134844A1
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dna
metallic oxide
fumed
solution
kit
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PCT/US2000/031005
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John Krupey
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Ligochem, Inc.
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Priority to EP00977161A priority Critical patent/EP1244811A1/fr
Priority to AU14836/01A priority patent/AU1483601A/en
Publication of WO2001034844A1 publication Critical patent/WO2001034844A1/fr

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    • 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

Definitions

  • the present invention relates to a method for isolating DNA from a proteinaceous medium such as whole blood, hemoglobin containing urine or saliva. Also disclosed are kits for practicing the method.
  • the method includes treating a specimen with a chaotropic agent such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA) ; or alternatively, heating the specimen in the presence of guanidine thiocyanate without the metal chelator being present, adding a protein precipitating agent and isolating the liquid phase, treating this liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated by flame hydrolysis in a solution of a metal salt which has weak charge attenuating properties, wherein the anion may be inorganic or organic.
  • a chaotropic agent such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA)
  • EDTA ethylene diamine tetraacetate
  • Nucleic acids are polymeric acids. In addition to having large numbers of nucleotides and ribose moieties, they possess a plurality of negatively charged phosphate groups. Because of their strong negative charge they should bind tightly to a positively charged metallic oxide surface. It has been demonstrated (Kummert R., and Strum ., International Journal of Colloid and Interface Science, 75(2) 373, 1980) that organic molecules with molecular masses smaller than 200 daltons and with the functional groups carboxylic, phenolic - OH or an amino group which can form covalent bonds with the structural metal, bind to the fumed aluminum oxide surface. The compounds that were employed in these studies were phthalic acid, benzoic acid, salicylic acid and catechol. Since the primary focus is on the binding of polymeric acids to the oxide surface, very little is to be gained from the studies which employed monomeric molecules.
  • the binding of a polyelectrolyte e.g. DNA
  • a surface containing multiple permanent charges of opposite sign is energetically more favorable than the binding of a single isolated monomeric unit (e.g. a deoxy ribonucleoside triphosphate) to the same surface.
  • a single isolated monomeric unit e.g. a deoxy ribonucleoside triphosphate
  • Silica is an oxide of the element Silicon. Silicon has properties between metals and non-metals and is called a metalloid.
  • Metallic oxides such as titanium oxide is an oxide of titanium which is a metal.
  • a metal is a substance having a characteristic luster, malleability and high electrical conductivity, that is, metals readily loose electrons to form positive ions.
  • a metal can be thought of as an array of nuclei immersed in a sea of electrons; some of the electrons present roam through the array of nuclei and acid and act as an all prevailing electrostatic glue. This is not the case with metalloids (silicon) where the electrons are less promiscuous and have a lesser tendency to wander about. All the atoms of metalloids are held together by a network of electron pair bonds.
  • silica particles When silica particles are placed in contact with water they do not acquire a permanent positive charge. Silica particles are mildly acid. Based on the experiments of Boom et al U.S. Patent No. 5,234,809, it appears that the interactive forces between the silica particles are weak in comparison to the strong electrostatic force that exists between the fumed metallic oxide and the nucleic acid since washing of the complex with pure water or neutral salt solutions tend to release significant amounts of nucleic acid from the surface. As a result of this property, Boom uses organic solvents to wash off extraneous proteins that are co- adsorbed onto the particles. Treating the nucleic acid-silica complex with an aqueous organic solvent to remove contaminating protein might be counterproductive, particularly if the protein is insoluble in that solvent composition.
  • a chaoptrope or chaotropic ion is a substance or anion which is least effective as a protein precipitant, and promotes unfolding, extension, and dissociation (Dandliker, W.B and de Saussure, V.A. in The Chemistry of Biosurfaces, Ed. M.L.Hair, Marcel Dekker, New York, 1971, pl8).
  • chaotropic anions are guanidine thiocyanate and potassium iodide .
  • kosmotropic ions are sodium chloride and sodium sulfate.
  • U.S. Patent No. 5,057,426 discloses a method for separating long chain nucleic acids comprising fixing the nucleic acids onto a porous matrix, washing the porous matrix to separate the other substances from the long chain nucleic acids, and removing the fixed long chain nucleic acids from the porous matrix.
  • the porous matrix is a material for chromatography having been modified with respect to its surface, and the material is based on a member selected from the group consisting of silica gel, diatomite, aluminum oxide, titanium oxide, hydroxylapatite, dextran, agarose, acrylamide, polystyrene, polyvinyl alcohol or other organic polymers, and derivatives or copolymers thereof.
  • United States Patent No. 5,470,463 relates to modified porous solid supports and processes for the preparation and use of same.
  • passivated porous mineral oxide supports are disclosed which are characterized by a reversible high sorptive capacity substantially unaccompanied by non-specific adsorption of or interaction with biomolecules .
  • Passivation is achieved by use of a passivation mixture comprising a main monomer, a passivating monomer and a crosslinking agent, which mixture upon polymerization results in the substantial elimination of the undesirable non-specific interaction with biomolecules.
  • United States Patent No. 5,599,667 discloses the use of polycationic solid supports in the purification of nucleic acids from solutions containing contaminants.
  • the nucleic acids non-covalently bind to the support without significant binding of contaminants permitting their separation from the contaminants.
  • the bound nucleic acids can be recovered from the support.
  • the supports as a means to separate polynucleotides and hybrids thereof with a nucleotide probe from unhybridized probe. Assays for target nucleotide sequences are described which employ this separation procedure.
  • United States Patent No. 5,635,405 discloses an aqueous colloidal dispersion for diagnostic or immunodiagnostic tests, comprising non-polymer nuclei surrounded by a hydrophilic copolymer that contains functional groups, a method for the detection of a specifically binding substance or immunochemically active component in a test fluid, and test kit containing the aqueous colloidal dispersion.
  • United States Patent No. 5,705,628 discloses a method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed.
  • the salt and polyalkylene glycol concentration of the solution is adjusted to levels which result in polynucleotide binding to the magnetic microparticles .
  • the magnetic microparticles with bound polynucleotides are separated from the solution and the polynucleotides are eluted from the magnetic microparticles.
  • the present invention provides a method for isolating DNA from a proteinaceous medium, such as whole blood, hemoglobin containing urine, or saliva, which comprises the steps of treating the specimen with a chaotropic agent, such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA) ; or alternatively heating the specimen in the presence of guanidine thiocyanate without the metal chelator being present, adding a protein precipitating agent and isolating the liquid phase, treating this liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated by flame hydrolysis in a solution of a metal salt which has weak charge attenuating properties, wherein the anion may be inorganic or organic.
  • a chaotropic agent such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA)
  • a suitable anion is chloride, and where it is organic, acetate may be used.
  • the procedure continues by separating the supernatant, washing the residue and removing the wash and then dissociating the DNA from the fumed alumina, titania, or zirconia by treatment with aqueous alkali borate, or phosphate or a metal hydroxide and recovering the liquid phase containing the DNA.
  • nucleated cell fraction There is also provided a method for isolating DNA from whole blood by first lysing the specimen and recovering the nucleated cell fraction.
  • the nucleated (white blood cells) are then treated with a chaotropic agent containing a chelator or treated with a chaotrope, and heated without the chelator being present.
  • the sample is treated with a precipitating agent and the supernatant that is recovered is processed in the same manner as described above.
  • the white blood cell fraction may be treated with a surfactant (e.g. 1.0% aqueous sodium dodecyl sulfate, SDS) containing the metal chelator EDTA, adding potassium acetate to neutralize the SDS, and to precipitate hemoglobin and isolating the liquid phase, treating this liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated by flame hydrolysis in a solution of a metal salt which has weak charge attenuating properties as described above. Further processing is achieved as described for the guanidine thiocyanate procedure.
  • a surfactant e.g. 1.0% aqueous sodium dodecyl sulfate, SDS
  • SDS sodium dodecyl sulfate
  • adding potassium acetate to neutralize the SDS
  • hemoglobin and isolating the liquid phase treating this liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated by
  • a method of isolating DNA from cells contained in a highly proteinaceous medium such as serum or plasma, or from a medium which has very little or no protein such as urine which comprises the steps of treating the medium with a chaotropic agent such as guanidine thiocyanate containing a metal chelator such as EDTA or alternatively, heating the specimen in the chaotropic agent in the absence of EDTA, and treating the liquid phase with either fumed alumina, fumed titania, or fumed zirconia followed by further processing as described.
  • the proteinaceous or non proteinaceous medium may be treated with a surfactant containing the metal chelator and treating the liquid phase with the metallic oxide, followed by further processing .
  • plasmids e.g. Bacterial Artificial Chromosome (BAC)
  • BAC Bacterial Artificial Chromosome
  • a fumed metallic oxide configuration consisting of non attenuated charges was prepared by dispersing the particles in ion free water. This preparation had a high binding capacity for RNA and a marginal binding capacity for DNA and can be employed to remove contaminating RNA from DNA containing preparations .
  • nucleic acid dispersed metallic oxide complexes generated are held together by strong ionic bonds contributed by multiply charged groups of opposite sign present on the respective interacting species. This situation presents a distinct advantage over the case that uses silica particles as adsorbent such as described in Boom, et al, supra. In the first place organic solvent need not be employed for washing procedure .
  • any buffer which contains an anion of unit negative charge such as a chloride, acetate and thiocyanate can be employed to thoroughly remove contaminating proteins from the dispersed metallic oxide complex without fear of desorbing the desired nucleic acid.
  • Divalent anions such as sodium dodecyl sulfate may also be employed.
  • Anions with a relatively high negative charge density such as phosphate, tetra-borate and citrate should not be employed.
  • Figure 1 shows a diagramatic representation of charged fumed metallic oxide particles dispersed in ion free water.
  • Figure 2 shows a diagramatic representation of attenuated charges on fumed metallic oxide particles dispersed in a sodium chloride solution.
  • Figure 3 shows the aggregation and sedimentation profiles of fumed metallic oxides in the presence and absence of DNA.
  • Figure 4 shows the electrophoretic patterns of DNA isolated from whole mouse blood and form urine spiked with white blood cells using fumed titanium oxide P25.
  • Figure 5 shows polymerase chain reaction (PCR) patterns of DNA isolated form whole mouse blood and from urine spiked with white blood cells using fumed titanium oxide P25.
  • Figure 6 shows polymerase chain reaction (PCR) of low copy number DNA in plasma which was captured by fumed titanium oxide P25 and subsequently released.
  • PCR polymerase chain reaction
  • Figure 7 shows the electrophoretic profiles of the BAC DNA isolated by the fumed metallic oxide process and the alcohol- centrifugation procedure.
  • Figure 8 shows an example of the isolation of DNA using the method of the present invention in a 96 well format.
  • Figure 9 shows a schematic of the method of the present invention .
  • the present invention relates to a method for isolating DNA from a proteinaceous medium such as whole blood, hemoglobin containing urine or saliva.
  • kits for practicing the method includes treating a specimen with a chaotropic agent such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA) , or alternatively heating the specimen in the presence of guanidine thiocyanate without the metal chelator being present; adding a protein precipitating agent and isolating the liquid phase; treating this liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated by flame hydrolysis in a solution of a metal salt which has weak charge attenuating properties, wherein the anion may be inorganic or organic.
  • a chaotropic agent such as aqueous guanidine thiocyanate containing a metal chelator such as ethylene diamine tetraacetate (EDTA)
  • a class of metal oxides produced by flame hydrolysis of the chlorides of the corresponding metals has been found to have broad-based applications in the fields of diagnostic and forensic medicine, as well as in the field of molecular bioinformatics .
  • These reagents which are the highly dispersed metallic oxides of aluminum, titanium, and zirconium, show a high degree of specificity for the nucleic acids, DNA and RNA, present in aqueous media (blood, urine, saliva, waste water) and will bind nucleic acid, irrespective of the volume of the sample.
  • the DNA metallic oxide complex is a white web-like structure consisting of a bridge work (i.e. a cross-linked structure) of DNA and metallic oxide. These aggregates settle rapidly under unit gravity.
  • RNA-metallic oxide complex is formed, and is recovered after centrifugation.
  • nucleic acid must be dissociated from the metallic oxide-nucleic acid complex in an intact form in order to permit its quantification and evaluation by current technologies (e.g. restriction enzyme digestion, polymerase chain reaction-PCR and sequencing) .
  • the three highly dispersed oxides of aluminum, titanium and zirconium which are used in the method of the present invention are sold under the trademarks Aluminum Oxide C, Titanium oxide P-25 and VP Zirconium Oxide, respectively, and are manufactured by Degussa.
  • the highly dispersed metallic oxides differ fundamentally from other oxides which are produced by precipitation, thermal decomposition or refinement of minerals.
  • the dispersed metallic oxides of aluminum, titanium, and zirconium have an average particle size of 13 nanometers, 21 nanometers and 30 nanometers respectively and a specific surface of 100m 2 /g, 50m 2 /g, and 40m 2 /g respectively.
  • the aluminum oxides produced through precipitation of aluminum hydroxide from aluminate solution followed by calcination consist of particles in the order of magnitude of micrometers.
  • High surface aluminum oxide gels, in contrast to fumed aluminum oxide (Aluminum Oxide C) have a high proportion of internal surface.
  • the dispersed metallic oxides have the pronounced tendency to remain electrostatically neutral in the powder condition.
  • the dispersed metallic oxide as exemplified by Aluminum Oxide C below, comes in contact with water, the substance of the solids binds protons and acquires a permanent positive net charge.
  • a chaotrope regardless of its concentration, whether it be high or low, is a suitable reagent for the dissociation of the nucleic and starting material and for the binding of the nucleic acid to the solid phase.
  • a chaotrope When present at lower concentrations (1.0-1.5 molar) a chaotrope may or may not exhibit chaotrope properties. It has been shown (Damonies, S., Int. J. Biol . Macromol . , 11,1, 1984) that chaotropes when present at a concentration in the range of 0.25-1.5 molar exert a stabilizing effect on bovine serum albumin.
  • the first series of binding experiments between nucleic acids (both DNA and RNA) and the dispersed metallic oxide surface were conducted in ion free water.
  • the reagents were (a) calf thymus DNA (Boehringer Mannheim, Germany) at a concentration of 50 micrograms per milliliter in water, (b) RNA from calf liver (Sigma Chemical Co., St.
  • the reagents were (a) calf thymus DNA at a concentration of 50 milligrams per milliliter in a 0.15 molar sodium chloride solution, (b) RNA from calf liver at a concentration of 50 milligrams per milliliter in a 0.15 molar sodium chloride, (c) dry fumed Aluminum Oxide C powder, (d) dry fumed Titanium Oxide-P25 powder, (e) a 2.5% weight/volume suspension of Aluminum Oxide C in 0.15 molar sodium chloride and, (f) a 2.5% weight/volume suspension of Titanium Oxide-P25 in 0.15 molar sodium chloride solution.
  • the pH of the respective nucleic acid solutions and the respective metallic oxide suspensions was adjusted to 7 by the addition of a few drops of a 0.1N sodium hydroxide solution .
  • Thirty-eight milligrams of the respective metallic oxide powders were added directly to one milliliter volumes of the respective nucleic acid solutions in sodium chloride.
  • One and one half milliliter volumes of the metallic oxide suspensions in sodium chloride were added to one milliliter volumes of the respective nucleic acids in the ionic medium.
  • the contents were processed as described above and the supernatants were analyzed for the presence or absence of the nucleic acids by UV absorption. The results are reported in Table 2.
  • RNA binding to the respective metallic oxide surfaces in an ion free medium was found to be greater than 90% on the particles that had been added in a dry form as well as for the oxide suspension in ion free water.
  • Fumed metallic oxides in suspension form in ion free water will not bind significant amounts of DNA when added to ion free aqueous solutions of DNA or to the DNA contained in a salt solution, but will bind large amounts of RNA when added to ion free aqueous solutions of RNA and to salt solutions containing RNA. Fumed metallic oxides in suspension form in salt solutions will bind large amounts of DNA and RNA when added to either ion free aqueous solutions or to salt solutions containing these two nucleic acids.
  • fumed metallic oxide particles acquire a strong positive charge when dispersed in water. This is an energetically unfavorable situation since it results in severe electrostatic repulsions between particles accompanied by an increase in the energy of thermal fluctuation. It was noted that when a suspension of either fumed aluminum oxide or fumed titanium oxide in deionized water was added to a solution of DNA in deionized water (Table 1) or to a solution of DNA in sodium chloride, (Table 3) very little or no metallic oxide DNA complex was formed. Since DNA binds to the metallic oxide surface by a bridging mechanism, this situation may be likened to "an attempt to construct a suspension bridge during a severe earthquake when the very foundations are rattling".
  • RNA degrading enzymes which are expensive and ion sensitive.
  • fumed metallic oxide suspensions in deionized water are cost effective and do not appear to be ion sensitive.
  • Inorganic salts Aqueous solutions of the sodium salts of the following anions: Chloride, sulfite, sulfate, bicarbonate, tetraborate and hydrogen phosphate were prepared at an ionic strength of 0.15.
  • Organic salts Aqueous solutions of the sodium salts of the following anions: Acetate, oxalate, and citrate were prepared at an ionic strength of 0.15.
  • Aluminum Oxide C (2.5 grams) and Titanium Oxide P25 (2.5 grams) were added to 100 milliliter volumes of the respective salt solutions to yield 2.5w/v % suspensions.
  • One and one haif-milliliter volumes of the metallic oxide suspensions were added to one milliliter volumes of the respective nucleic acids.
  • the tubes were mixed by inversion for about 10 seconds and then centrifuged at 10,000 x g for 5 minutes. The supernatants were recovered and analyzed for the presence or absence of DNA by UV absorption at 260 and 280 nanometers .
  • the chloride and acetate ions appear to be the preferred anionic species for optimal charge attenuation since maximal DNA binding (92.4% to Al oxide, 90.4% to Ti oxide for chloride, and 90.2% to Al oxide, 90.0% to Ti oxide for acetate) to the surface occurs in their presence. There seems to be some marginal correlation between the number of units of negative charge present on the different organic carboxylate ions and ability to neutralize the oxide surface. The effectiveness increases in the order citrate 3" oxalate' " > acetate 1" as evidenced by an increase in DNA binding with decreasing charge.
  • the valence of the species is not the only factor that is responsible for charge attenuation.
  • Anions with the same number of unit charges exhibit strikingly different activities when exposed to the metallic oxide surface.
  • sulfate which has 2 units of negative charge is much less effective than hydrogen phosphate (HP0 4 2 ⁇ ) and tetraborate (B 4 0 7 2" ) in neutralizing the positive charge on the metallic oxide surface. This is evidenced by the fact that less DNA is bound in the presence of HP0 4 : ⁇ and B 4 0 7 2" than in the presence of SO,, 2" .
  • Electronegativity is the property of an atom describing its ability to attract an electron pair.
  • Oxygen is the most electronegative and is assigned a value of 3.45, followed by Nitrogen (2.98), Carbon (2.55), Sulfur (2.53), Hydrogen (2.13) and Phosphorus (2.10) and Boron (2.0).
  • the negative charge density surrounding the oxygen atoms involved in the O-P bond in the HP0 4 2 ⁇ ion or the oxygen atoms involved in the B 4 0 7 2 ⁇ ion would be greater than the negative charge density surrounding the oxygen atoms in the 0-S bond in the S0 4 2 ⁇ ion or any of the other counterions used in the present invention. Therefore, surface charge attenuation by anions is largely dependent on the chemical nature of the atoms that constitute the ionic species.
  • Aluminum oxide C(2.5 grams), titanium oxide P25 (2.5 grams ) and VP zirconium oxide (2.5 grams) were added to 100- milliliter volumes of sodium chloride, ionic strength 0.15, to yield 2.5w/v % suspensions.
  • One and one half-milliliter volumes of the metallic oxide suspensions were added to one- illiliter volumes of DNA containing 50 micrograms of DNA per
  • the pellets were then separately treated with 1.0 0 milliliter volumes of the following solutions: 0.02M tribasic sodium phosphate pH 9.2, 0.02M sodium tetra-borate pH 9.5 and 0.02M sodium hydroxide pH 12.0.
  • the DNA recoveries are reported in Table 5.
  • the urine sample is collected and guanidine thiocyanate added to the sample to a final concentration of 3M.
  • Guanidine thiocyanate is a chaotropic agent, and functions by disrupting the cell and the nucleohistone complex, and thereby releasing free DNA into the aqueous media.
  • the solution is then diluted with water to a final concentration of 1.5M with respect to guanidine thiocyanate.
  • a suspension of metallic oxide in 0.15M sodium chloride is then added to form an aggregate of metallic oxide and DNA. The aggregate is then allowed to settle to the bottom of the tube. Alternatively the tube can be subjected to low speed centrifugation and the aggregate recovered as a tight pellet.
  • the DNA-metallic oxide complex is washed three times with deionized water after which it is dissociated with 0.02M solution of sodium hydroxide and neutralized with a 0.1M solution of Tris HC1.
  • organic solvents e.g. phenol, chloroform, isopropanol
  • the following protocol is employed for this purpose. In this procedure, one volume of whole blood is treated with two volumes of a chaotropic agent such as 3M guanidine thiocyanate in a buffer, say, 100 mM sodium acetate pH 7.0.
  • ProCipitateTM manufactured by Ligochem Inc., Fairfield NJ
  • the composition of ProCipitate is disclosed in U.S. Patent Nos. 5,294,681; 5,453,493; and 5,534,597, and U.S. Application Serial No. 08/676,668 (now allowed) incorporated herein by reference in their entireties.
  • the tubes are then centrifuged at 10,000 x g for 15 minutes, and the supernatant recovered, 1.5 volumes of Titanium Oxide P-25 is then added.
  • the resulting aggregate consisting of DNA and metallic oxide is allowed to settle under unit gravity. After settling the supernatant is removed by aspiration and the settled complex is washed with three washings using deionized water.
  • the tubes are then centrifuged at 1000 x g for 30 seconds. The supernatant is discarded and 0.02M sodium hydroxide is added to the tube.
  • the tubes are then vortexed, followed by centrifugation at, say, 10,000 x g for 5 minutes.
  • the supernatants are then removed neutralized with a 0.
  • IM Tris HCl solution and analyzed for DNA by spectrophotometric absorption at 260 and 280 nm.
  • One ml of whole blood contains approximately 40 to 50 micrograms of DNA. This quantity translates into about one absorbance unit (AU) at 260 nm and 0.8 AU at 280 nm.
  • the DNA specimens are also subjected to agarose gel electrophoresis in which the DNA bands were identified by ethidium bromide staining.
  • ProCipitateTM is added to precipitate the protein.
  • the supernatant is recovered by centrifugation and this DNA containing solution is processed and analyzed for DNA as described above.
  • one volume of whole blood is treated with three volumes of a 1.0% w/v of sodium dodecyl sulfate (SDS) in a buffer, say, 10 mM solution of Tris buffer and lOOmM EDTA pH 8.0. After remaining at room temperature for 15 minutes, 3 volumes of a 3M solution of potassium acetate is added to neutralize the SDS and to precipitate the hemoglobin that is present. The tubes are then centrifuged and the supernatant is recovered. 1.5ml of Titanium Oxide P-25 suspension is then added.
  • SDS sodium dodecyl sulfate
  • Titanium Oxide P25 were dispersed in 300ml of 0. IM sodium hydroxide. The suspension was gently stirred for 30 minutes and then allowed to settle under unit gravity. The supernatant was removed by aspiration and discarded. The settled oxide was then treated with 300ml of 0.15M sodium chloride and mixed for 5 minutes. After settling, the supernatant was removed. The process of sodium chloride addition, mixing, followed by settling under unit gravity was repeated two more times. To the oxide was added 300ml of a 10w/v% solution of polyethylene glycol 8000 (Mw 7000-8000 daltons) in 0.9% sodium chloride. After thorough dispersion followed by mixing for 30 minutes the suspension was allowed to settle and the supernatant was discarded.
  • Mw 7000-8000 daltons polyethylene glycol 8000
  • the sediment was then washed with two 300ml volumes of 0.9% NaCl to insure complete removal of weakly bound polyethylene glycol.
  • the oxide was dispersed in 250ml of 0.9% sodium chloride, the pH was adjusted to 7 by the slow addition of 0.1N HCL and the volume was adjusted to 300ml by the addition of 0.9% sodium chloride to yield a 3.0 w/v% suspension.
  • the surface area of the Titanium Oxide P25 particle ranges from 60-70 m/g, the average particle diameter is 30 nanometers.
  • Dissociation Buffer A solution of 0.02M sodium hydroxide in deionized water.
  • Neutralizing Buffer A solution of 0. IM Tris (hydroxymethyl) amino methane hydrochloride (Tris HCl) pH .8 in deionized water.
  • SDS sodium dodecyl sulfate
  • the lysing solution consisted of 1 part of a solution containing 0.9% sodium chloride (saline) and 2 parts water.
  • the tubes were mixed by repeated inversion for 1.0 minute, and then centrifuged at 2000 x g for 10 minutes. The supernatant which consisted mainly of lysed red blood cells and other serum components was removed by aspiration and discarded.
  • the pellet which consisted of white blood cells and hemoglobin dispersed in 1.0ml of saline and recentrifuged in order to remove contaminating hemoglobin, and then resuspend in 1.0 ml of saline.
  • DNA does not generally occur in a free form, but is bound instead to the histone fraction in nucleated cells.
  • High concentrations of guanidine thiocyanate have the property of disrupting the nucleohistone complex with the concomitant release of DNA into solution.
  • the aggregate was then washed further with 2x10 milliliter portions of distilled water with repeated inversion within a period of 5 minutes. After the final wash, the tube was centrifuged at 2000 x g for 5 minutes, and the residual liquid was removed by aspiration. One ml of sodium hydroxide solution (0.02M pH12.0) was added to the pellet, and followed by repeated mixing with a micropipette in order to insure complete dissociation of DNA from the metallic oxide surface. The preparation was then centrifuged at 10,000 x g for 6 minutes. DNA was then recovered in the supernatant. The alkaline supernatant was acidified to pH 8.0 by the addition of 150 microliters of 0.1N Tris HCl.
  • the absorbance at 260nanometers was 0.90 and the ebsorbance at 280 nanometers was 0.52. These values translate into a 260:280 ratio of 1.73 with a yield of about 90%.
  • K e kilobases
  • ProCipitateTM 200 microliters was then added to the dispersed pellet followed by vortexing for 10 seconds. The tube was then centrifuged at 10,000 x g for 5 minutes after which time the supernatant was recovered. Titanium Oxide suspension (150 microliters) was then added to the DNA containing supernatant. The resulting aggregate consisting of DNA and titanium oxide was allowed to settle under unit gravity. After settling, the supernatant was removed by aspiration and 1.0 milliliter of deionized water was added to the aggregate in order to remove contaminating protein and residual guanidine thiocyanate . The contents were mixed by gentle inversion, after which the supernatant was discarded.
  • the aggregate was then washed further with 2 x 10 milliliter portions of deionized water by repeated inversion. After the final wash, the tube was centrifuged at 3000 x g for 5 minutes and the residual liquid was removed by aspiration. Eighty microliters of 20 millimolar (mM) sodium hydroxide was added to the pellet, and followed by repeated mixing with a micropipette in order to insure complete dissociation of DNA from the metallic oxide surface. The preparation was then centrifuged at 10,000 x g for 6 minutes. DNA was then recovered in the supernatant. The alkaline supernatant was acidified to pH 8.0 by the addition of 10 microliters of 0. IM Tris HCl. The absorbance at 260 nm was 0.76 and the absorbance at 280 nano-meters was 0.43. These values translate into a 260:280 ratio of 1.77 with a yield of 76%.
  • mM millimolar
  • Potassium acetate 100 microliters was then added to the solubilized pellet, followed by vortexing for 10 seconds. The tube was then centrifuged at 10,000 x g for 5 minutes after which the supernatant was recovered. Titanium Oxide suspension (150 microliters) was then added to the DNA containing supernatant. After settling and removal of the supernatant, the aggregate was washed with three 500 microliter portions of deionized water, followed by removal of the supernatant after each wash. Further processing was achieved as described for the guanidine thiocyanate- ProCipitateTM procedure.
  • the biological sample is first extracted with a suitable reagent that will release both DNA and RNA into the aqueous medium.
  • Fumed metallic oxide particles, that have been previously dispersed in deionized water are added to the treated sample to adsorb RNA.
  • the mixture is then centrifuged, and the supernatant containing the DNA is recovered.
  • Fumed metallic oxide particles that have been previously dispersed in a salt solution are then added to capture DNA.
  • the DNA-metallic oxide complex is then washed, and dissociated to release free DNA, followed by neutralization.
  • the lysing solution consisted of 1 part of a solution containing 0.9% sodium chloride and 2 parts water.
  • the tubes were mixed by inversion, allowed to stand at room temperature for 10 minutes, and then centrifuged at 2000 x g for 10 minutes.
  • the supernatant which consisted mainly of lysed red blood cells and other serum components, was removed by aspiration and discarded.
  • the pellet consisted of white blood cells and large amounts of residual blood components.
  • the quantity of hemoglobin in the pelleted fraction was found to be 7.0 milligrams.
  • the urine specimen was prepared by adding 0.3 grams of lyophilized male urine (Sigma) to 9.0 ml of distilled water. This solution was then added to the white blood cell pellet and inverted several times to insure proper mixing. Dissociation of the nucleohistone complex to release free DNA DNA does not generally exist in a free form, but is bound instead to the histone fraction in nucleated cells. Dissociation of the nucleic acid-histone complex is achieved by treatment with a chaotropic reagent. This treatment results in cell disruption and dissociation of the DNA histone complex to release free DNA into the solution.
  • Contaminating proteins were removed by treating the chaotrope containing urine with a water insoluble cross-linked polymeric acid, trade name ProCipitateTM. This product is described in US Patents Nos . 5,294,681/5,453,493 and
  • the tube was gently inverted, and the aggregate consisting of nucleic acid complexed to the oxide was allowed to settle under gravity. The supernatant was carefully aspirated and discarded. The pellet was washed twice with 500 microliter portions of deionized water. After the final wash, the pellet was centrifuged at 3000 x g for 5 minutes, and the residual liquid was removed. The DNA was dissociated from the complex by thoroughly dispersing the pellet in 40 microliters of 20 millimolar (mM) sodium hydroxide using a micropipet. The suspension was then centrifuged through a 0.2 micrometer membrane filter at 3000 x g for. 10 minutes. The filtrate containing DNA was then neutralized with 5 microliters of 0. IM Tris HCl.
  • mM millimolar
  • the absorbance at 260 nm was 0.65 and the absorbance at 280 nano-meters was 0.36. These values translate into a 260:280 ratio of 1.8 with a yield of 65%. After electrophoresis in agarose gels, a single sharp fluorescent band was observed with a molecular mass greater than 20 kilobases (kb) .
  • the DNA was also subjected to repeated amplification cycles using the Polymerase Chain Reaction (PCR) and was found to have the same properties as highly purified DNA.
  • the isolation of DNA from whole blood may be performed by heating the sample in the presence of guanidine thiocyanate in the absence of the metal chelator EDTA.
  • the procedure was performed as follows: To 50 microliters of whole mouse blood was added 100 microliters of a 3M solution of guanidine thiocyanate in 0. IM sodium acetate pH 7.0. The solution was then vortexed and incubated at 65°C for 10 minutes. After cooling to room temperature, 250 microliters of ProCipitateTM was added. The sample was then processed using the same procedure that was employed for the isolation of DNA in the presence of EDTA using room temperature conditions and which is described above.
  • the absorbance at 260 nm was 0.70 and the absorbance at 280 nanometers was 0.38. These values translate into a 260:280 ratio of 1.8 with a yield of 70%.
  • a single band was observed with a molecular mass greater than 20 kilobases (kb) .
  • the DNA was found to be suitable for amplification by Polymerase Chain Reaction (PCR) .
  • PCR Polymerase Chain Reaction
  • the product after amplification was subjected to agarose gel electrophoresis using the conditions described above.
  • the electrophoretic profile is shown in Figure 5.
  • the pellet was washed two more times with DEPC treated water. The pellet was then treated with 40 microliters of a 0.02M solution of sodium hydroxide followed by mixing with a micropipet using up and down motion to dissociate the complex. The contents was transferred to a microfilter (0.22um) centrifuge tube and spun at 3,000 x g for 10 minutes. To the filtrate was added 7ul of 0.1N Tris HCl pH 4.8. Two microliters of this preparation was amplified by the Polymerase Chain Reaction (PCR) using the reagents and amplification protocol of Qiagen (Taq PCR Hand Book, Page 12, May 1997). Human ⁇ actin primer was purchased from Perkin Elmer Catalog ⁇ : N808-0230, Foster-City, California; the size of the amplicon generated is 294 bp. The following cycle program was used:
  • the product after amplification was subjected to agarose gel electrophoresis using the conditions described above.
  • the electrophoretic profile is shown in Figure 6.
  • BAC Bacterial Artificial Chromosome. These are large circular constructs of double stranded DNA similar to plasmids but many times larger. BACs are on the order of 120 to 300 kb in diameter as compared with 2kb for regular plasmids.
  • E. coli containing the BAC construct were grown in Luria Broth (LB) as described by Kelley J.M. et al . (Nucleic Acids Research, 27, 1539-1546, 1999) .
  • BAC culture Thirty milliliters of the BAC culture were centrifuged at 1500 x g for 10 minutes. The supernatants were decanted and the bacterial pellet was dispersed in 1.25 milliliters of lOmM Tris buffer containing 5.0mM EDTA. Seventy-five microliter aliquots of the bacteria suspension were subsequently employed in all studies. Seventy-five microliters of lysis solution (1.0% sodium dodecyl sulfate in 0.2N sodium hydroxide) was added to each tube. The tubes were mixed by inversion and allowed to stand at room temperature for about 5 minutes.
  • a kit for use in a method for isolating deoxyribonucleic acid (DNA) from other substances in solution includes, comprises, consists of or consists essentially of (A) a substrate for holding a specimen, (B) a chaotrope, (C) a protein precipitating agent, (D) an adsorbent comprising a fumed metallic oxide, and (E) a DNA dissociating agent an example of a chaotrope is guanidine thicyanate.
  • the chaotrope may include guanidine thicyanate and a buffer.
  • the protein precipitating agent is preferably ProcipitateTM.
  • the DNA dissociating agent is preferably a 0.02 molar solution of sodium hydroxide.
  • the adsorbent is preferably a fumed metallic oxide selected from the group consisting of aluminum oxide, titanium oxide, and zirconium oxide.
  • the fumed metallic oxide may be used in combination with an aqueous solution of sodium salts of an anion selected from the group consisting of acetate, bicarbonate, sulfate, sulfite, chloride, oxalate, and citrate.
  • the fumed metallic oxide is preferably used in combination with 0.15M NaCl.
  • the fumed metallic oxide may be used in combination with deionized water.
  • the dissociating agent is preferably selected from the group consisting of 0.02M P0 4 2" , 0.02M B 4 0 2" and OH 1" .
  • the dissociating agent may alternatively be sodium hydroxide.
  • the kit may further comprise a neutralizing buffer.
  • the neutralizing buffer may be TrisHCl in deionized water.
  • the adsorbent may be fumed metallic oxide.
  • the adsorbent includes a fumed metallic oxide and is attached to a solid-phase carrier.
  • the solid-phase carrier can be a plastic bead or a microtiter plate.
  • the solid-phase carrier may be composed of insoluble polymers.
  • the solid-phase carrier may be a polymer of styrene or vinyl chloride or a copolymer thereof.
  • the solid phase carrier may take any form, such as a container, a stick or bead.
  • kits and process of the present invention can advantageously be used in forensic medicine, molecular bioinformatics, in an automated systems for the isolation of bacterial and viral constructs for genomic sequencing, for non-invasive diagnostics, capture and quantification of DNA in saliva-capture, quantification of small quantities of DNA present in large volumes of urine, and removal of contaminating nucleic acids in the downstream processing of recombinant proteins.
  • compositions, kits, and methods are described as including or comprising specific components, it is contemplated by the inventors that compositions of the present invention also consist essentially of, or consist of the recited components.

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Abstract

L'invention concerne une méthode permettant d'isoler un ADN d'un milieu protéique tel que du sang total, de la salive ou de l'urine contenant de l'hémoglobine. L'invention concerne également des trousses de test permettant de réaliser cette méthode.
PCT/US2000/031005 1999-11-10 2000-11-13 Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode WO2001034844A1 (fr)

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EP00977161A EP1244811A1 (fr) 1999-11-10 2000-11-13 Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode
AU14836/01A AU1483601A (en) 1999-11-10 2000-11-13 Method for isolating dna from a proteinaceous medium and kit for performing method

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WO2003104251A3 (fr) * 2002-06-07 2004-07-15 Dna Genotek Inc Compositions et procedes permettant d'obtenir des acides nucleiques a partir des expectorations
WO2008033936A3 (fr) * 2006-09-12 2008-07-24 Sierra Molecular Corp Elimination d'interferences sur des dosages moleculaires d'acides nucleiques mettant en œuvre des solutions tamponnees de chaotropes
EP1992689A1 (fr) * 2006-02-15 2008-11-19 Tosoh Corporation Procede d'extraction d'acide nucleique a partir d'un materiau biologique
WO2008113017A3 (fr) * 2007-03-14 2008-11-27 Sierra Molecular Corp Compositions, systèmes et procédés de conservation et/ou de stabilisation d'une cellule et/ou d'une macromolécule
US8158357B2 (en) 2005-03-16 2012-04-17 Dna Genotek Inc. Compositions and method for storage of nucleic acid from bodily fluids
US8221381B2 (en) 2005-12-09 2012-07-17 Dna Genotek Inc. Container system for releasably storing a substance
CN112321870A (zh) * 2020-11-06 2021-02-05 浙江工商大学 一种微针贴片及其制备方法和应用
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
WO2021160849A1 (fr) 2020-02-14 2021-08-19 Enalees Kits et procédés d'extraction d'acides nucléiques à partir de kits d'échantillons complexes et procédés d'extraction d'acides nucléiques à partir d'échantillons complexes
US11284867B2 (en) 2019-06-20 2022-03-29 Spectrum Solutions L.L.C. Sample collection system including a sample collection vessel, sealing cap, and reagent chamber and valve assembly in the sealing cap
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US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11655495B2 (en) 2017-01-16 2023-05-23 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use
US11712692B2 (en) 2018-11-20 2023-08-01 Spectrum Solutions L.L.C. Sample collection system including sealing cap and valve
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US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci

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US10619187B2 (en) 2002-06-07 2020-04-14 Dna Genotek Inc. Compositions and methods for obtaining nucleic acids from sputum
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