[go: up one dir, main page]

WO1992018514A1 - Purification d'acides nucleiques a l'aide de supports en oxyde m tallique - Google Patents

Purification d'acides nucleiques a l'aide de supports en oxyde m tallique Download PDF

Info

Publication number
WO1992018514A1
WO1992018514A1 PCT/US1992/002262 US9202262W WO9218514A1 WO 1992018514 A1 WO1992018514 A1 WO 1992018514A1 US 9202262 W US9202262 W US 9202262W WO 9218514 A1 WO9218514 A1 WO 9218514A1
Authority
WO
WIPO (PCT)
Prior art keywords
metal oxide
dna
nucleic acid
nucleic acids
particles
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.)
Ceased
Application number
PCT/US1992/002262
Other languages
English (en)
Inventor
Chan-Wha Kim
Eric F. Funkenbusch
Kyung H. Moh
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.)
3M Co
Original Assignee
Minnesota Mining and Manufacturing Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Minnesota Mining and Manufacturing Co filed Critical Minnesota Mining and Manufacturing Co
Publication of WO1992018514A1 publication Critical patent/WO1992018514A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to methods and kits for the purification of nucleic acids, and more particularly, to insoluble supports usefui in such methods, and to kits incorporating such supports.
  • nucleic acids can involve, for instance, either the purification of one or more types of nucleic acids from other types of nucleic acids, or from other cellular materials.
  • Conventional methods for nucleic acid purification include the use of ultracentrifugation, phenol extraction, and the like. See, e.g., Basic Methods in Molecular Biology. L.G. Davis et al., eds., Elsevier, NY, 1986.
  • Plasmids are small, generally circular, pieces of nucleic acid that can be modified by recombinant techniques to include gene sequences coding for proteins of interest. In the course of developing and using such "recombinant" plasmids it is highly desirable to be able to purify the plasmids from all other cellular constituents, including from cellular genomic (e.g., chromosomal) DNA.
  • nucleic acid purification methods have been described that involve the use of a solid support, e.g., for the immobilization of the desired nucleic acid, or for the removal of the unwanted cellular components.
  • U.S. Patent No. 4,923,978 describes the use of a rehydrated silica gel capable of binding enzymes so as to leave nucleic acids unbound.
  • U.S. Patent No. 4,935,342 describes a method for isolating or purifying nucleic acids that involves the use of particular anion exchange materials and control of the molarity of the various solutions used.
  • 4,648,975 describes silicon- based chromatographic and reactive materials with surfaces modified to contain or to be coated with oxides, hydrous oxides, hydroxides, carbonates, or silicates of aluminum, iron, or other suitable metals such as zirconium or titanium.
  • the modified support can be used as a chromatographic support for separating nucleosides, nucleotides, and nucleic acids.
  • European Patent Publication 0 391 608 describes metal oxide supports and compositions of metal oxides and nucleic acids that are useful, for example, in methods for the hybridization of nucleic acids.
  • the present invention provides a method for the purification of nucleic acids comprising the steps of
  • nucleic acid that sorbs to the support is or includes the desired nucleic acid.
  • nucleic acid that sorbs can include undesired types of nucleic acid(s) , in such a manner that the desired nucleic acid thereby remains in solution in a more purified form, at least insofar as it has now been separated from the sorbed nucleic acid.
  • the present invention provides a kit for the purification of nucleic acids.
  • the kit includes means for lysing cells in suspension in order to release substantially all nucleic acid into solution, together with a metal oxide support capable of sorbing released nucleic acid.
  • Supports of the present invention if sorbed with a desired nucleic acid, are useful in a variety of ways, e.g., as chromatographic columns. Such supports provide a particular advantage in that the sorbed nucleic acids can thereafter be readily desorbed at will.
  • the method also includes the steps of washing the sorbed support, then desorbing and eluting the nucleic acid.
  • the method is useful for any biological or other sample containing the desired nucleic acid, e.g., where the desired nucleic acid is present in combination with unwanted biomolecules such as other cellular components.
  • the method can be used, for instance, for the purification of a desired nucleic acid from eucaryotic cells, or from procaryotic organisms such as animal and bacterial cells, yeast cells, and viruses.
  • FIG. 1 depicts a flow diagram for the purification of plas id DNA from a cell suspension.
  • FIG. 2 depicts a flow diagram for the recovery of fragmented DNA from a gel.
  • FIG. 3 is a graph depicting sorption of calf thymus DNA on zirconia particles.
  • FIG. 4 is a graph depicting sorption kinetics of calf thymus DNA on zirconia particles in deionized water.
  • FIG. 5 is a graph depicting sorption kinetics of calf thymus DNA on alumina particles in deionized water.
  • FIG. 6 is a graph depicting selective sorption of calf thymus DNA on alumina particles from a mixture with bovine serum albumin ("BSA”) .
  • FIG. 7 is a graph depicting recovery yield of DNA from a mixture with BSA using zirconia particles.
  • FIG. 8 is a graph depicting recovery yield of RNA from a mixture with BSA using alumina particles.
  • the present invention discloses that metal oxide supports, such as those described in European
  • Patent Publication 0 391 608 can also be used to purify all, or a desired type, of the nucleic acids, e.g., from a cell suspension or other solution or from another support, such as a gel.
  • the method is adaptable to the purification of any desired nucleic acid, including linear or circular plasmid DNA, chromosomal DNA, or RNA.
  • the word “purify”, and inflections thereof, refers to the separation of one physical or chemical type of nucleic acid from one or more other types within a sample such as a biological sample, such as DNA from RNA, or circular plasmid DNA from chromosomal DNA.
  • the word also refers to the separation of nucleic acids from non-nucleic acid biomolecules such as the proteins, lipids, and other components making up the sample.
  • the purification of nucleic acids according to this invention can therefore include both the purification of nucleic acids from complex solutions such as cell lysates and cell extracts, as well as the clarification of nucleic acid-containing solutions, such as those prepared in the course of in vitro syntheses of nucleic acids.
  • purity is reflected in three alternative ways, taking into account either: (1) the recovery yield of nucleic acid, i.e., the amount (e.g., mass) of recovered nucleic acid divided by the total amount of the same nucleic acid present in the sample; (2) the relative purity, i.e., the amount of the nucleic acid recovered divided by the amount of other nucleic acids and/or biomolecules in the sample; and (3) the biological activity of the recovered nucleic acid, i.e., the ability of the nucleic acid to function in the desired biological manner.
  • nucleic acid(s) refers to deoxyribonucleic acid ("DNA”) and ribonucleic acid (“RNA”) , including various nucleic acid “types” such as circular or linear plasmid DNA and RNA, genomic DNA and RNA, and fragmented DNA and RNA (e.g., nucleic acid fragments that have been separated as one or more bands in a gel) .
  • the term therefore refers to any nucleic acid type or combination of nucleic acid types that are biochemically or physically separable from other types or combinations, e.g., by preferential precipitation, conventional sorption techniques, or gel separation.
  • FIG. 1 there is outlined a presently preferred method for the purification of plasmid DNA from a cell suspension.
  • FIG. 2 there is outlined a presently preferred method for the purification of separated, fragmented DNA from a gel band.
  • the preferred method begins with the release from procaryotic cell culture 12 of substantially all nucleic acids from the cell into solution, e.g., by lysing or extracting the cells to create cell extract 14.
  • Any suitable procaryotic, eucaryotic, or other suspension can be used, as long as it contains the desired nucleic acid.
  • the cells can be lysed or extracted in any manner, e.g., chemical, physical, and/or biological that is suitable to release into solution at least the desired type(s) of nucleic acid.
  • the cells are portrayed as being completely lysed, thereby releasing not only the desired plasmid DNA, but substantially all other cellular components as well. Lysis in this particular case is preferably accomplished by the addition of an alkaline lysing solution sufficient to increase the pH of the sample to between about pH 12 and about pH 13.
  • the alkaline lysing solution also preferably includes sodium dodecyl sulfate ("SDS”), in order to precipitate cellular RNA and protein.
  • SDS sodium dodecyl sulfate
  • the lysing solution is added in an amount, concentration, and under conditions suitable to provide the desired extent of lysis of the cells.
  • the lysing solution is then "neutralized", i.e., further lysis is terminated in order to allow chromosomal DNA, large molecular weight RNA, and protein to be precipitated.
  • an alkaline lysing solution as described above can be neutralized by potassium acetate. See, e.g., H.C. Birnboim, Meth. Enz ⁇ mol. - 100:243-255 (1983).
  • the unwanted RNA is substantially removed from solution by any suitable means within the skill of those in the art, e.g., by the use of an RNase enzyme capable of digesting the RNA, or, preferably, by lithium chloride (LiCl) precipitation (18) of the RNA.
  • the solution is subjected to an alcohol (e.g., isopropanol) precipitation (16) prior to LiCl treatment.
  • the alcohol precipitates large RNA and plasmid DNA, leaving smaller RNA in solution.
  • the pellet can then be resuspended in fresh buffer and the LiCl used to precipitate the large RNA alone, thereby leaving plasmid DNA in solution.
  • the desired nucleic acid which in the present diagram would still be substantially intact and in solution, is sorbed (20) to a metal oxide support, which in turn is then removed from the solution, thereby leaving other impurities, such as proteins and salts, in solution.
  • sorb and inflections thereof, is used interchangeably with the word “adsorb” herein, and refers to the attachment of nucleic acid to a metal oxide support by physical and/or chemical interactions when the nucleic acid and support are combined in solution according to the method of the present invention.
  • the sorbed nucleic acid can thereafter be washed (22) and, if desired, desorbed and eluted (24) as described more fully below.
  • FIG. 2 depicts a flow diagram (30) , discussed more fully in the EXAMPLES below, for the recovery of fragmented DNA from a gel.
  • Suitable metal oxides of the present invention exhibit an optimal combination of such properties as strength of sorption and sorption capacity, with respect to nucleic acids.
  • metal oxide refers collectively to metal oxides and hydroxides as well as hydrous metal oxides and hydroxides as that term is defined and used in European Patent Publication 0 391 608.
  • hydrous metal oxides and hydroxides as that term is defined and used in European Patent Publication 0 391 608.
  • hydroous refers to metal oxide or hydroxide surfaces containing physically and/or chemically adsorbed water, and will be used in parentheses herein to indicate the optional presence of such water.
  • “Strength” of sorption refers to the ability of sorbed nucleic acid to remain sorbed to the metal oxide for purposes of the intended use of the resulting composition.
  • “Sorption capacity” of the metal oxide refers to the ability of the particular metal oxide support to sorb enough nucleic acid, per unit weight or surface area of the support, to purify the solution and/or for the subsequent intended use of the resulting composition. In turn, it relates to the amount of nucleic acid that a particular metal oxide can sorb per unit weight or surface area.
  • suitable metal oxides include, but are not limited to, the (hydrous) oxides and hydroxides of: calcium, cobalt, hafnium, iron, lanthanum, magnesium, manganese, nickel, titanium, yttrium, and zinc.
  • preferred metal oxides include those of iron, cobalt, and nickel.
  • Examples of preferred metal oxides include, but are not limited to, the oxides and hydroxides of zirconium and aluminum, including in particular zirconia (Zr0 2 ) and alumina (A1 2 0 3 ) .
  • Zr0 2 zirconia
  • A1 2 0 3 alumina
  • compositions that are of predominantly the alpha alumina form.
  • Alumina particles can be prepared by methods known to those skilled in the art, for example by spray drying or atomizing. See, e.g., U.S. Patent No. 4,931,414. In order to obtain particles having desired and reproducible particle diameter distribution and size, it is preferred to prepare such particles by an emulsion technique such as that described further below.
  • the accessible (i.e., to the desired nucleic acid(s)) surface area of a support is greater than about 1 m 2 /g metal oxide, especially about 3 m 2 /g, and particularly preferred are supports having surface areas of greater than about 5 m 2 /g metal oxide.
  • Surface area can be determined in any suitable fashion. For purposes of the present invention, surface area is determined by nitrogen absorption, as described in Absorption Surface Area and Porosity. S.J. Gregg and K.S.W. Sing, Academic Press, London and New York (1967) .
  • Metal oxide can be formed into any suitable support.
  • metal oxide particles can be used in suspension or packed in a column when the nucleic acid is to be sorbed from aqueous suspension, or in the form of a colloidal particle suspension, coating, or composite structure (e.g., membrane) incorporating particles.
  • a colloidal particle suspension, coating, or composite structure e.g., membrane
  • the particle size will vary depending on the form of the support, e.g., when packed in a column, metal oxide particles having an average diameter of about 5 ⁇ m to about 500 ⁇ m will generally be used, and preferred will be particles of about 10 ⁇ m to about 200 ⁇ m.
  • metal oxide particles having an average diameter of about one-tenth ⁇ m to about 50 ⁇ m will generally be used, and preferred are particles of about one ⁇ m to about 10 ⁇ m.
  • metal oxide particles having an average diameter of about 1 ⁇ m to about 500 ⁇ m will generally be useful, with particles about 10 ⁇ m to about 100 ⁇ m being preferred.
  • the purified nucleic acid can be used for any suitable purpose, e.g., if still sorbed to a support the nucleic acid can be used in that form, for instance, as a chromatographic or otherwise reactive column.
  • the support with sorbed nucleic acid can be washed to remove nonspecifically bound proteins and salts, e.g., with water or sodium or potassium chloride solutions.
  • the plasmid DNA can be desorbed, eluted and recovered from the support with a suitable elution buffer, e.g., by the use of Tris(hydroxymethy1)aminomethane hydrochloride (“Tris") - ethylenediamine tetraacetic acid (“EDTA”) buffer (e.g. , 5 to 30 mM Tris and 0 to 5 mM EDTA and about pH 7 to about pH 9) or phosphate buffer (e.g., 10 to 50 mM and about pH 6 to about pH 8) .
  • the present invention also provides a kit for the purification of nucleic acids.
  • the kit incorporates both means for lysing a cell suspension as well as a metal oxide support capable of sorbing the released nucleic acid.
  • Lysing means can include any suitable physical, chemical, and/or biological (e.g., enzymatic) method.
  • lysing means include the combination of a lysis buffer to suspend the cells, together with an alkaline lysis reagent in order to actually disintegrate (i.e., rupture or lyse the cells), and a neutralizing solution in order to precipitate the desired nucleic acid (e.g., chromosomal DNA).
  • a suitable lysis buffer is a combination of Tris (e.g., 10 mM to 50 mM) : EDTA (e.g., 1 mM to 20 mM) : glucose (e.g., 0 mM to 100 mM) in solution.
  • a suitable alkaline lysis reagent is the combination of base, such as NaOH on the order of about 0.1 N to about 0.5 N, together with a surfactant such as sodium dodecyl sulfate ("SDS”) , on the order of about one-tenth to about two percent by weight, based on the weight of the reagent.
  • a suitable neutralizing solution is potassium acetate, e.g., on the order of 2 Molar to 4 Molar.
  • Optimum Concentration of Metal Oxide Particles A 4500 g sample of "Nyacol Zr 100/20" a colloidal Zr ⁇ 2 sol manufactured by Nyacol Inc., Ashland, Massachusetts and containing 20% (by weight) of Zr0 2 particles primarily of about 100 nm in size was concentrated on a rotary evaporator until its concentration was about 35% Zr0 2 by weight. This sol was then spray dried on a spray dryer manufactured by Nyro Inc. About 900 g of dried spherical particles were obtained ranging in size from about 0.5 ⁇ m to about 30 ⁇ m. The dried zirconia particles were heated in a furnace to a temperature of 600°C over 6 hours and held at 600°C for 6 more hours.
  • the furnace was then turned off and allowed to cool.
  • the resulting fired spherical Zr0 2 particles were then air classified and fractions from about 1 ⁇ m to about 5 ⁇ m or about 10 ⁇ m to about 20 ⁇ m in diameter were used in subsequent experiments.
  • plot C represents 200 g zirconia/g DNA and plot D represents 400 g zirconia/g DNA.
  • plot E represents 4 g alumina/g DNA and plot F represents 20 g alumina/g DNA. Therefore, recovery of DNA by sorption on the metal oxide particles is sufficiently rapid to allow its use in routine bioseparation processes.
  • alumina particles such as those described in EXAMPLE 4 (400 g alumina/g DNA) were added. The mixtures were shaken for 10 minutes and centrifuged for 5 minutes at 14,000 rpm. The supernatant was sampled and the absorbance of the samples was measured at 260 and 280 nm to determine the DNA and protein remaining. The adsorption of DNA and BSA was determined from the concentration difference in the supernatant.
  • FIG. 6 shows the effects of NaCl on the selective sorption of DNA on alumina particles.
  • Plot G represents DNA and plot H represents BSA.
  • concentration of NaCl is higher than about 200 mM, greater than 99% of DNA in the solution sorbed on the alumina particles, leaving greater than 90% of BSA in solution. Therefore, alumina particles selectively sorbed DNA from the mixture. Such selective absorption was also demonstrated using zirconia particles.
  • (D) alumina particles having high alpha alumina were prepared by an emulsion technique as described below.
  • Particles (D) were prepared in the following manner:
  • a sol precursor "A” was prepared from 0.019 g of magnesium nitrate (reagent grade magnesium nitrate hexahydrate, MCB Manufacturing Chemicals, Inc., Cincinnati, OH) dissolved in 59.7 grams of colloidal alumina sol (NalcoTM #614 10% solids in aqueous solution, Nalco Chemical Co., Napierville, IL) ;
  • X-ray diffraction was performed using an automatic powder diffractometer (Philips "APD 3600", Philips Electronic Instruments, Inc. , Mahwah, NJ) .
  • the identified metal oxide phases were the predominant species and are listed in decreasing order of peak intensity.
  • the particles (A) were predominantly gamma alumina with a minor amount of theta alumina and trace amounts of beta aluminum hydroxide.
  • the particles (B) were predominantly eta alumina with minor amounts of beta aluminum hydroxide.
  • the particles (C) were predominantly alpha alumina with minor amounts of beta aluminum hydroxide and trace amounts of theta alumina, and the particles (D) were predominantly alpha alumina with minor amounts of beta alumina, and were substantially devoid of aluminum hydroxide.
  • Surface area was determined by conventional Brunauer-Em ett-Teller ("BET”) nitrogen adsorption technique using a QuantasorbTM model "SW-6" surface area measuring instrument (Quantachrome Corp. Syosset, NY) . Density was determined by helium pycnometry using a Quantachrome Stereo Pycnometer (Quantachrome Corp.). Size and shape were determined by visual examination using transmission light microscopy.
  • each of the particles exhibited significant adsorption of plasmid DNA.
  • the somewhat lower adsorption of particles (C) may be due to their larger particle size (average about 44 ⁇ m diameter) .
  • the alumina particles (D) exhibited substantially total recovery under the particular conditions employed.
  • EXAMPLE 5 Desorption of DNA Sorbed on Metal Oxide Particles Calf thymus DNA (0.005%) was sorbed on zirconia particles prepared as described in EXAMPLE 1 (400 g particles/g DNA) from a mixture with BSA (0.05%) at 200 mM NaCl in the manner described in EXAMPLE 3. After centrifugation the supernatant was discarded and the pellet was resuspended in 200 mM NaCl to wash out non-specifically bound BSA. The samples were centrifuged for 5 minutes at 14,000 rpm and the supernatant was discarded. The pellets were resuspended in increasing concentrations of potassium phosphate (pH 7.0) and shaken for 30 minutes.
  • potassium phosphate pH 7.0
  • the mixtures were centrifuged for 5 minutes at 14,000 rpm.
  • the supernatants were sampled and absorbance at 260 and 280 nm were measured to determine the concentrations of DNA and BSA.
  • the recovery yield of DNA and BSA was determined as the concentration difference from the initial concentration as in EXAMPLE 4.
  • EXAMPLE 6 Desorption of RNA Sorbed on Metal Oxide Particles Baker's yeast RNA (0.03% weight/volume) was sorbed to alumina particles (2 ⁇ m average diameter, 0.17 g particles/mg RNA) prepared according to the method described in EXAMPLE 4 from a mixture with BSA (0.05%) at 400 mM NaCl in a manner such as that described in EXAMPLE 3. The RNA sorbed on alumina particles was desorbed using potassium phosphate in the manner as described in EXAMPLE 5.
  • EXAMPLE 7 Purification of Plasmid DNA from E. coli with Metal Oxide Particles Plasmid pUCll ⁇ was purified from E. coli JM83 (described in Gene. 19:259 (1982)) with alumina particles (7 ⁇ m in diameter) prepared as described in EXAMPLE 4, following the procedure shown in FIG. 1. Ten ml of E. coli JM83 culture in a 15 ml centrifuge tube was spun down and the supernatant was discarded. Cells were suspended in 1 ml of deionized water and transferred to a microcentrifuge tube. The cells were again spun down and the supernatant was discarded.
  • the cells were resuspended in 0.2 ml of "TGE" buffer (25 mM Tris-HCl, 50 mM glucose, 10 mM EDTA, pH 8.0).
  • the resuspended cells were lysed by adding 0.4 ml of alkaline lysis reagent (0.2 N NaOH, 1% SDS) and neutralized with 0.4 ml of neutralizing solution (3 M potassium acetate, pH 6.5). Isopropanol (0.6 to 0.7 ml) was added to the tube to precipitate plasmid DNA from the neutralized cell lysate. The tube was mixed for 1 minute and centrifuged for 1 minute. The supernatant was discarded and the pellet was dissolved in 0.2 ml of deionized water.
  • a 90% saturated lithium chloride solution in deionized water (0.4 ml, prepared by combining 9 volumes of a saturated solution with 1 volume water) was added and mixed for 15 seconds to precipitate RNA.
  • the tube was spun for 1 minute.
  • the supernatant was transferred to a new microcentrifuge tube and centrifuged again for 1 minute.
  • the supernatant was transferred into a new microcentrifuge tube.
  • the alumina particles (30 to 50 ⁇ l of 40% suspension in water, pH 2.0 - 4.0) were added into the supernatant of the lithium chloride precipitation, mixed for 15 seconds, and spun for 5 seconds.
  • the supernatant was discarded and the pellet was washed twice with 0.4 ml of deionized water.
  • the plasmid was eluted in 100 ⁇ l of "TE" buffer (10 mM Tris-HCl, 3 mM EDTA, pH 8.0) by incubating for 5 minutes in a 50°C to 60°C water bath
  • the purification of plasmid DNA from the cell pellet was accomplished within 30 minutes without using conventional CsCl gradient ultracentrifugation or phenol extraction.
  • the recovery yield of the plasmid DNA was more than 80%, which was estimated by comparing the intensity of the electrophoresis gel bands.
  • the absorbance ratio (260 nm/280 nm) of the plasmid DNA recovered was higher than 1.93, thereby indicating that the DNA was substantially pure with respect to protein.
  • plasmid DNA recovered was incubated for 18 hours in a 37°C water bath. The incubated plasmid was run on gel and no DNase activity was found.
  • EXAMPLE 8 Purification of Plasmid DNA from Cells with Metal Oxide Minicolumns Plasmid pUC118 was purified from E. coli JM83 with an alumina minicolumn (7 ⁇ m average particle diameter, prepared according to the method described in EXAMPLE 4) by following the procedure shown in the diagram of FIG. 1. E. coli in 40 ml culture was spun down and resuspended in 4 ml of TGE buffer. The resuspended cells were lysed by adding 4 ml of alkaline lysis buffer and neutralized with 4 ml of neutralizing solution. The neutralized cell lysate was centrifuged and the supernatant was transferred into a new 50 ml centrifuge tube.
  • the centrifuge tube was spun for 1 minute and the supernatant was loaded on the alumina minicolumn (0.5 g particles in a 4 ml column).
  • the column was washed twice with 1 ml of 50% ethanol.
  • the plasmid was eluted with 1 ml of TE buffer or potassium phosphate buffer (50 mM, pH 7.0).
  • Plasmid pUCll ⁇ was digested with restriction enzyme Taq I for 2 hours in a 55°C water bath.
  • the digested plasmid (1.2 to 2.3 ⁇ g) was run on a 1.5% agarose gel in order to separate the various fragment sizes formed.
  • the DNA was recovered from the gel by following the procedure shown in the diagram of FIG. 2.
  • the DNA band was excised using a razor blade and was placed in a microcentrifuge tube.
  • the gel slice was melted (i.e., liquefied) by adding 2 to 3 volumes of a gel liquefaction solution, in this case 6 M Nal, although about 4 M to about 8 M Nal would be suitable, and incubating for 2 minutes in a 50°C to 60°C water bath.
  • a gel liquefaction solution in this case 6 M Nal, although about 4 M to about 8 M Nal would be suitable, and incubating for 2 minutes in a 50°C to 60°C water bath.
  • a gel liquefaction solution in this case 6 M Nal, although about 4 M to about 8 M Nal would be suitable
  • To sorb DNA from the gel melt 1 ⁇ l to 10 ⁇ l of 40% alumina particle suspension (7 ⁇ m average diameter, prepared as according to the method of EXAMPLE 4) was added, mixed for 5 to 8 minutes, and the tube centrifuged for 15 seconds. The pellet was washed twice with 0.5 ml of 50% ethanol.
  • Glass Particle I was GlassmilkTM particles, obtained from BiolOl, San Diego, CA
  • Glass Particle II was US BiocleanTM particles obtained from United States Biochemical, Cleveland, OH.
  • results are provided in TABLE 4 below, where recovery values were determined as described below.
  • the recovery yield of DNA with alumina particles was as high as 70% of the total DNA added into the gel, while the recovery yield with glass particles was only 50%.
  • the recovery yield with alumina particles (higher than 50%) was again much higher than that of the glass particles (30% with glass particle I and 10% with glass particle II) . Therefore, the use of alumina particles provides significant advantages, especially when the amount of DNA is limited.
  • Plasmid pUC118 prepared according to the method described in Example 4 above was purified with an alumina-loaded EmporeTM membrane.
  • the alumina-loaded EmporeTM membrane was prepared according to the general method of Example 2 of U.S. Pat. No. 4,906,378. Briefly stated, 25 g of alumina particles (18 ⁇ m average diameter, prepared according to the method described in EXAMPLE 4) was combined with 4.6 g of polytetrafluoroethylene resin emulsion (TeflonTM 30B available from E. I.
  • E. coli in 10 ml culture was spun down and resuspended in 0.2 ml of TGE buffer.
  • the resuspended cells were lysed by adding 0.4 ml of alkaline lysis buffer and neutralized with 0.4 ml of neutralizing solution.
  • the neutralized cell lysate was centrifuged and the supernatant was transferred into a new microcentrifuge tube.
  • To the supernatant 0.7 ml of isopropanol was added, mixed until a precipitate formed, and centrifuged. The supernatant was discarded and the pellet was dissolved in 0.2 ml of water.
  • a 90% saturated lithium chloride solution (0.4 ml) was added to the centrifuge tube.
  • the centrifuge tube was mixed and spun for 1 minute.
  • the supernatant was loaded on the spin-filter unit containing 1 to 4 layers of the alumina-loaded membrane.
  • the spin-filter unit was centrifuged for 3 minutes at 6,000 rpm.
  • the membrane was washed twice with 0.4 ml of water by spinning the micro spin-filter unit for 2 minutes at 6,000 rpm.
  • Plasmid DNA was desorbed and eluted with 0.1 ml of TE buffer by spinning the micro spin-filter unit for 2 minutes at 6,000 rpm.
  • the amount of plasmid DNA recovered was more than 22 ⁇ g when two or more layers of membranes were used.
  • the absorbance ratio (260 nm/280 nm) of the plasmid DNA recovered was higher than 2.1. This indicated that the plasmid DNA recovered was nearly 100% pure (with respect to protein) .
  • the purified plasmid DNA was also able to transform E. coli at a frequency equivalent to plasmids purified by both the commercial process or conventional CsCl gradient centrifugation, achieving a rate of approximately 10 6 transformants per microgram plasmid) . No detectable damage to the lac Z gene encoded within the plasmid was observed.
  • DNA purified from an agarose gel according to the method described in EXAMPLE 9 was used in the cloning of 1.6 kb Bam HI generated fragment containing Corvnebacterium glutamicum aro A gene.
  • the purified DNA (both the vector and target fragments) was able to form recombinant molecules at an efficiency equivalent or better than that of control DNA purified according to the instructions of a commercial (GENECLEANTM) process.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Saccharide Compounds (AREA)

Abstract

Procédé et trousses permettant de purifier des acides nucléiques, dans lesquels on utilise des supports en oxyde métallique pouvant absorber de manière préférentielle l'acide nucléique présent dans une solution. On peut ensuite effectuer la désorption et l'élution de l'acide nucléique absorbé, ou bien l'utiliser tel quel à l'état absorbé.
PCT/US1992/002262 1991-04-12 1992-03-20 Purification d'acides nucleiques a l'aide de supports en oxyde m tallique Ceased WO1992018514A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US68512591A 1991-04-12 1991-04-12
US685,125 1991-04-12

Publications (1)

Publication Number Publication Date
WO1992018514A1 true WO1992018514A1 (fr) 1992-10-29

Family

ID=24750876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/002262 Ceased WO1992018514A1 (fr) 1991-04-12 1992-03-20 Purification d'acides nucleiques a l'aide de supports en oxyde m tallique

Country Status (2)

Country Link
AU (1) AU1978692A (fr)
WO (1) WO1992018514A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2753204A1 (fr) * 1996-09-11 1998-03-13 Transgene Sa Procede de preparation d'adn plasmidique
EP0818461A3 (fr) * 1996-07-12 1999-02-10 Toyo Boseki Kabushiki Kaisha Procédé pour isoler des acides ribonucléiques.
JP3082908B2 (ja) 1996-07-12 2000-09-04 東洋紡績株式会社 リボ核酸の単離方法
WO2001034844A1 (fr) * 1999-11-10 2001-05-17 Ligochem, Inc. Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode
WO2001046404A1 (fr) 1999-12-22 2001-06-28 Abbott Laboratories Methode et kit d'isolement pour acide nucleique
EP0897978A3 (fr) * 1997-08-22 2001-10-17 Becton, Dickinson and Company Oxyde de zirconium et de composés apparentés pour la purification des acides nucléiques
US6329515B1 (en) * 1998-09-11 2001-12-11 Jin Ho Choy Bio-inorganic compound capable of stable, solid-state storage of genes and preparation thereof
US6383783B1 (en) 1999-09-21 2002-05-07 3M Innovative Properties Company Nucleic acid isolation by adhering to hydrophobic solid phase and removing with nonionic surfactant
WO2004005306A2 (fr) 2002-07-04 2004-01-15 Technische Universität Dresden Objet metallique recouvert d'acides nucleiques et de derives d'acides nucleiques, son procede de production et d'utilisation
DE10237518A1 (de) * 2002-08-16 2004-02-26 Süd-Chemie AG Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien
EP1003908A4 (fr) * 1997-04-16 2004-05-12 Immunological Associates Of De Archivage d'acides nucleiques
WO2005007852A3 (fr) * 2003-07-09 2005-06-09 Genvault Corp Elution d'acides nucleiques a temperature ambiante
US7208271B2 (en) 2001-11-28 2007-04-24 Applera Corporation Compositions and methods of selective nucleic acid isolation
EP1432818A4 (fr) * 2001-08-31 2007-05-30 Applera Corp Archivage d'acides nucleiques
US7727710B2 (en) 2003-12-24 2010-06-01 3M Innovative Properties Company Materials, methods, and kits for reducing nonspecific binding of molecules to a surface
US20100207051A1 (en) * 2006-12-21 2010-08-19 Invitrogen Dynal As Particles and their use in a method for isolating nucleic acid or a method for isolating phosphoproteins
US7939249B2 (en) 2003-12-24 2011-05-10 3M Innovative Properties Company Methods for nucleic acid isolation and kits using a microfluidic device and concentration step
US7955801B2 (en) * 2006-04-05 2011-06-07 Samsung Electronics Co., Ltd. Method and apparatus for disrupting cells and purifying nucleic acid using a single chip
KR20170128392A (ko) 2015-03-20 2017-11-22 도레이 카부시키가이샤 핵산의 회수 방법
WO2018052011A1 (fr) 2016-09-14 2018-03-22 東レ株式会社 Procédé de récupération d'adn acellulaire
EP3322805A4 (fr) * 2015-07-14 2019-01-23 Abbott Molecular Inc. Purification d'acides nucléiques à l'aide d'oxydes de titane-cuivre

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3652761A (en) * 1969-09-04 1972-03-28 Corning Glass Works Immunochemical composites and antigen or antibody purification therewith
DD230560A3 (de) * 1983-11-07 1985-12-04 Leiser Robert Matthias Verfahren zur isolierung und partiellen reinigung von rna
US4648975A (en) * 1983-08-17 1987-03-10 Pedro B. Macedo Process of using improved silica-based chromatographic supports containing additives
EP0268946A2 (fr) * 1986-11-22 1988-06-01 DIAGEN Institut für molekularbiologische Diagnostik GmbH Méthode de séparation d'acides nucléiques à longues chaînes
EP0389063A2 (fr) * 1989-03-23 1990-09-26 Akzo Nobel N.V. Procédé de purification d'acides nucléiques
EP0391608A2 (fr) * 1989-04-03 1990-10-10 Minnesota Mining And Manufacturing Company Supports d'oxydes métalliques pour des acides nucléiques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3652761A (en) * 1969-09-04 1972-03-28 Corning Glass Works Immunochemical composites and antigen or antibody purification therewith
US4648975A (en) * 1983-08-17 1987-03-10 Pedro B. Macedo Process of using improved silica-based chromatographic supports containing additives
DD230560A3 (de) * 1983-11-07 1985-12-04 Leiser Robert Matthias Verfahren zur isolierung und partiellen reinigung von rna
EP0268946A2 (fr) * 1986-11-22 1988-06-01 DIAGEN Institut für molekularbiologische Diagnostik GmbH Méthode de séparation d'acides nucléiques à longues chaînes
EP0389063A2 (fr) * 1989-03-23 1990-09-26 Akzo Nobel N.V. Procédé de purification d'acides nucléiques
EP0391608A2 (fr) * 1989-04-03 1990-10-10 Minnesota Mining And Manufacturing Company Supports d'oxydes métalliques pour des acides nucléiques

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0818461A3 (fr) * 1996-07-12 1999-02-10 Toyo Boseki Kabushiki Kaisha Procédé pour isoler des acides ribonucléiques.
US5990302A (en) * 1996-07-12 1999-11-23 Toyo Boseki Kabushiki Kaisha Method for isolating ribonucleic acid
JP3082908B2 (ja) 1996-07-12 2000-09-04 東洋紡績株式会社 リボ核酸の単離方法
WO1998011208A1 (fr) * 1996-09-11 1998-03-19 Transgene S.A. Procede de preparation d'adn plasmidique
FR2753204A1 (fr) * 1996-09-11 1998-03-13 Transgene Sa Procede de preparation d'adn plasmidique
EP1003908A4 (fr) * 1997-04-16 2004-05-12 Immunological Associates Of De Archivage d'acides nucleiques
EP0897978A3 (fr) * 1997-08-22 2001-10-17 Becton, Dickinson and Company Oxyde de zirconium et de composés apparentés pour la purification des acides nucléiques
US6329515B1 (en) * 1998-09-11 2001-12-11 Jin Ho Choy Bio-inorganic compound capable of stable, solid-state storage of genes and preparation thereof
US6383783B1 (en) 1999-09-21 2002-05-07 3M Innovative Properties Company Nucleic acid isolation by adhering to hydrophobic solid phase and removing with nonionic surfactant
US6790642B2 (en) 1999-09-21 2004-09-14 3M Innovative Properties Company Method for reducing the amount of nucleic acid adhering to a hydrophobic surface
WO2001034844A1 (fr) * 1999-11-10 2001-05-17 Ligochem, Inc. Methode permettant d'isoler un adn d'un milieu proteique et trousse utilisee pour realiser cette methode
US6936414B2 (en) * 1999-12-22 2005-08-30 Abbott Laboratories Nucleic acid isolation method and kit
EP1240320B1 (fr) * 1999-12-22 2011-06-15 Abbott Laboratories Methode et kit d'isolement pour acide nucleique
WO2001046404A1 (fr) 1999-12-22 2001-06-28 Abbott Laboratories Methode et kit d'isolement pour acide nucleique
EP1432818A4 (fr) * 2001-08-31 2007-05-30 Applera Corp Archivage d'acides nucleiques
US7537898B2 (en) 2001-11-28 2009-05-26 Applied Biosystems, Llc Compositions and methods of selective nucleic acid isolation
US8507198B2 (en) 2001-11-28 2013-08-13 Applied Biosystems, Llc Compositions and methods of selective nucleic acid isolation
US8865405B2 (en) 2001-11-28 2014-10-21 Applied Biosystems Llc Compositions and methods of selective nucleic acid isolation
US7208271B2 (en) 2001-11-28 2007-04-24 Applera Corporation Compositions and methods of selective nucleic acid isolation
WO2004005306A2 (fr) 2002-07-04 2004-01-15 Technische Universität Dresden Objet metallique recouvert d'acides nucleiques et de derives d'acides nucleiques, son procede de production et d'utilisation
DE10237518A1 (de) * 2002-08-16 2004-02-26 Süd-Chemie AG Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien
WO2005007852A3 (fr) * 2003-07-09 2005-06-09 Genvault Corp Elution d'acides nucleiques a temperature ambiante
US7727710B2 (en) 2003-12-24 2010-06-01 3M Innovative Properties Company Materials, methods, and kits for reducing nonspecific binding of molecules to a surface
US7939249B2 (en) 2003-12-24 2011-05-10 3M Innovative Properties Company Methods for nucleic acid isolation and kits using a microfluidic device and concentration step
US8658359B2 (en) * 2006-04-05 2014-02-25 Samsung Electronics Co., Ltd. Method and apparatus for disrupting cells and purifying nucleic acid using a single chip
US20110183325A1 (en) * 2006-04-05 2011-07-28 Samsung Electronics Co., Ltd. Method and apparatus for disrupting cells and purifying nucleic acid using a single chip
US7955801B2 (en) * 2006-04-05 2011-06-07 Samsung Electronics Co., Ltd. Method and apparatus for disrupting cells and purifying nucleic acid using a single chip
US20100207051A1 (en) * 2006-12-21 2010-08-19 Invitrogen Dynal As Particles and their use in a method for isolating nucleic acid or a method for isolating phosphoproteins
KR20170128392A (ko) 2015-03-20 2017-11-22 도레이 카부시키가이샤 핵산의 회수 방법
US11118173B2 (en) 2015-03-20 2021-09-14 Toray Industries, Inc. Method of collecting a nucleic acid(s)
US11015187B2 (en) 2015-07-14 2021-05-25 Abbott Molecular Inc. Purification of nucleic acids using copper-titanium oxides
US10392613B2 (en) 2015-07-14 2019-08-27 Abbott Molecular Inc. Purification of nucleic acids using copper-titanium oxides
US10526596B2 (en) 2015-07-14 2020-01-07 Abbott Molecular Inc. Purification of nucleic acids using metal-titanium oxides
EP3322805A4 (fr) * 2015-07-14 2019-01-23 Abbott Molecular Inc. Purification d'acides nucléiques à l'aide d'oxydes de titane-cuivre
EP3988658A1 (fr) * 2015-07-14 2022-04-27 Abbott Molecular Inc. Purification d'acides nucléiques à l'aide d'oxydes de cuivre-titane
US11608496B2 (en) 2015-07-14 2023-03-21 Abbott Molecular Inc. Purification of nucleic acids using copper-titanium oxides
US12234448B2 (en) 2015-07-14 2025-02-25 Abbott Molecular Inc. Purification of nucleic acids using titanium oxides
KR20190049717A (ko) 2016-09-14 2019-05-09 도레이 카부시키가이샤 무세포 dna의 회수 방법
WO2018052011A1 (fr) 2016-09-14 2018-03-22 東レ株式会社 Procédé de récupération d'adn acellulaire

Also Published As

Publication number Publication date
AU1978692A (en) 1992-11-17

Similar Documents

Publication Publication Date Title
WO1992018514A1 (fr) Purification d'acides nucleiques a l'aide de supports en oxyde m tallique
JP4594467B2 (ja) 常磁性粒子を用いた核酸の精製とその操作方法
JP3253638B2 (ja) シリカ磁気粒子を使用する生物学的目標物質の分離法
AU751324C (en) Solid-phase nucleic acid isolation
EP1266385B1 (fr) Particules de verre ferro- ou ferrimagnetiques poreuses servant a isoler des molecules
JP2703636B2 (ja) 核酸を精製する方法
US8288169B2 (en) Surface mediated self-assembly of nanoparticles
US8569477B2 (en) Method for isolating nucleic acids comprising the use of ethylene glycol multimers
US20070026435A1 (en) Hydroxysilane functionalized magnetic particles and nucleic acid separation method
US20020106686A1 (en) Methods and reagents for the isolation of nucleic acids
EP1173559A1 (fr) Procede de purification d'acides nucleiques a l'aide de carbure de silicium
JP2002502856A (ja) 核酸の単離および精製方法
JP2008134239A (ja) 磁性色素
EP1071695A1 (fr) Reduction d'endotoxines dans la purification d'acides nucleiques
JP2002543835A (ja) 常磁性粒子を用いた集細胞及びライセート清澄化
CA2294749C (fr) Dispositif et procede de concentration et purification de molecules biologiques en une etape
JPH11196869A (ja) リボ核酸の単離方法
RU2382081C2 (ru) Способ выделения и очистки нуклеиновых кислот
AU772552B2 (en) Methods of isolating biological target materials using silica magnetic particles
MXPA98007681A (en) Methods of isolating biological target materials using silica magnetic particles

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA