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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
  • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
  • B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D2221/00—Applications of separation devices
  • B01D2221/10—Separation devices for use in medical, pharmaceutical or laboratory applications, e.g. separating amalgam from dental treatment residues

Definitions

• the present invention relates to a method for the purification of extrachromosomal DNA using a flow-through centrifuge.
• Plasmid DNA is generally expressed in cells, e.g. B. amplified in gram-negative bacteria, especially in E. coli. The cells are then disrupted and the plasmid DNA isolated therefrom. The isolated plasmid DNA can then be used for molecular biological or medical applications, e.g. B. for the construction of cloning vectors, for the transformation of prokaryotic and for the transfection of eukaryotic cells.
• Various methods are known for disrupting the cells and for obtaining the plasmid DNA (see J. Sambrook et al., 5 Molecular Cloning, A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press).
• WO92 / 12780 describes a technical design of a flow-through centrifuge and its use for the separation of macromolecule mixtures.
• Four standard proteins, for example, are separated in an aqueous 2-phase system at a maximum of 1000 rpm in accordance with the respective distribution coefficients of the proteins. Due to the different elution times, the components of the mixture are obtained separately.
• the present invention relates to a method for the purification of extrachromosomal DNA, which is characterized in that an extrachromosomal DNA and liquid containing further cell components is passed under conditions through a flow-through centrifuge which lead to the separation of the extrachromosomal DNA from insoluble cell components and the purified extrachromosomal DNA is obtained.
• flow centrifuges have so far been used exclusively for cell separation. It has now surprisingly been found that flow centrifuges can also be used to purify extrachromosomal DNA in large quantities without the extrachromosomal DNA being damaged by the shear forces that occur. Also surprising was the fact that the chromosomal DNA present in the suspension of the disrupted cells is not fragmented during the flow centrifugation and can thus be separated quantitatively from the extrachromosomal DNA.
• the extrachromosomal DNA which is purified by the method according to the invention, can be linear or circular, single or double-stranded.
• the DNA is preferably a circular and double-stranded plasmid DNA.
• the cell containing extrachromosomal DNA can be a prokaryotic or eukaryotic cell, preferably it is a bacterial cell, in particular a gram-negative cell, such as an E. coli cell. If necessary, cells can be used which contain so-called artificial chromosomes as extrachromosomal DNA.
• Artificial chromosomes are linear double-stranded DNA molecules that are generally called YAC (yeast artificial chromosome) and are amplified in yeast cells.
• the liquid containing extrachromosomal DNA used in the method according to the invention is preferably a cell lysate.
• the cell lysate is particularly preferably produced by alkaline lysis of cells containing extrachromosomal DNA and subsequent acidification. But it can also other common cell bursting methods are used, such as combinations of enzyme (lysozyme) and heat treatment.
• Any amount of cellular biomass can be used as the starting material for the method according to the invention.
• a biomass of 100 g to 50 kg is preferably lysed per batch.
• the liquid containing extrachromosomal DNA is generally introduced into the flow-through centrifuge by gradient or / and pumping.
• a flow-through centrifuge with a volume adapted to the lysis approach is used in the method according to the invention.
• a volume of at least 0.1 to 50 1, particularly preferably of 0.2 to 4 1, is preferably used.
• the centrifuge container is preferably cylindrical.
• the flow-through centrifuge is operated at a suitable g number, preferably at 10,000 to 40,000 x g. Examples of commercially available flow centrifuges are "CEPA" high-speed centrifuges or high-performance centrifuges from Carr (USA), which currently have a capacity of up to 9,000 l / h.
• the process according to the invention is generally carried out continuously.
• the suspension of the lysed biomass is introduced into the flow-through centrifuge from below.
• the rotation of the centrifuge tube (10,000 - 40,000 x g) separates solid components such as cell wall components and genomic DNA adhering to them from the wall of the centrifuge tube.
• the purified solution containing extrachromosomal DNA generally emerges from the top of the flow-through centrifuge, although it is also conceivable for the solution containing extrachomosomal DNA to escape laterally, downward or elsewhere.
• the flow-through centrifuge can be operated at different temperatures, preferably at 4 ° C carried out to room temperature.
• extrachromosomal DNA of different sizes can be purified, preferably extrachromosomal DNA with a size of 1 kbp to 200 kbp is purified.
• the extrachromosomal DNA is preferably linear, circular or supercoiled plasmid DNA.
• the extrachromosomal DNA can be further purified. For example, in order to remove RNA from the solution, an RNase treatment is carried out. Furthermore, chromatographic purification steps, such as, for example, anion exchange chromatography, affinity chromatography or hydroxylapatite chromatography, can also be carried out.
• suitable materials for anion exchange chromatography are organic or inorganic polymers and copolymers, such as, for example, polymethacrylate (Macroprep-Biorad, Germany), polystyrene-divinylbenzene (Poros-Perseptive, HyperD-Biosepra, Source-Pharmacia) or silica gel, positive on the surface thereof invited groups z. B.
• Diethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) - groups are bound.
• a particularly preferred material for anion exchange chromatography is Q-Sepharose.
• a particularly preferred material for affinity chromatography is hydroxylapatite.
• the DNA solution obtained can be subjected to cross-flow filtration for further purification, concentration and / or buffering.
• cross-flow filtration extensive removal of endotoxins from the DNA preparation can also be achieved.
• the DNA solution is guided tangentially past one or more semipermeable membranes, the exclusion size of which is chosen such that the DNA molecules are retained by the membranes and substances with a lower molecular weight can pass through the membranes, an endotoxin-free DNA Solution is obtained.
• the extrachromosomal DNA obtained by the process according to the invention is essentially undamaged and has essentially no single or double strand breaks.
• a plasmid DNA purified according to the invention after gel electrophoretic separation shows only one dominant band which corresponds to the "covalently closed circle” conformation. Furthermore, in addition to the bands corresponding to the "Open Circle” and “Linearized Circle” conformations, there are no other bands.
• the DNA obtained by the method according to the invention can easily be used for conventional molecular biological and medical applications, such as for cloning, for transformation, for transfection, for microinjection into cells, for use in methods of gene therapy, DNA vaccination and / or for polymerase.
• Chain reaction (PCR) can be used.
• the present invention relates to the use of a flow-through centrifuge for the purification of extrachromosomal DNA.
• a CEPA laboratory centrifuge LE (open design) with a clarification cylinder made of stainless steel (1.4571, V4A) is used in the test carried out. About 2,000 g of biomass are digested using the alkali lysis method (modified method according to Birnboim & Bolybo Birnboim & Doly, Nucl. Acid Res. 7 (1979) 1513-1523).
• the viscous suspension is pumped through the inlet opening into the flow-through centrifuge.
• the centrifuge is operated with a g-number of 10,000-18,000 x g. As soon as escaping liquid becomes cloudy, the precipitate has to be removed from the cylinder and centrifugation has to be continued after inserting the cleaned cylinder.
• the clear plasmid DNA solution freed from the cellular impurities emerges from the top of the flow-through centrifuge and is collected in a vessel.
• chromatography is carried out on Q-Sepharose and hydroxylapatite.
• the decanted centrifuge supernatant is adjusted to 49 - 50 mS / cm conductivity and to 5 + 4 ° by adding TE buffer (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA pH 8.5 ⁇ 0.2) C cooled. All chromatography is carried out at this temperature. The centrifugation supernatant is drawn onto the equilibrated column. The column is then washed with about 8 SV 10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.65 mol / 1 NaCl pH 8.5 + 0.2.
• a gradient (5 SV buffer A (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.65 mmol / 1 NaCl pH 8.0 ⁇ 2), 5 SV buffer B (10 mmol / 1 Tris-HCl, 1 mmol / 1 EDTA, 0.85 mol / 1 NaCl pH 8.0 + 0.2)) and the eluate is fractionated, the detection takes place at 254 nm.
• the prepeak (impurities) becomes the main peak (Plasmid DNA) separated by collecting the main peak from the rising flank in a separate vessel.
• Equilibration buffer 0.1 mol / 1 potassium phosphate, 6 mol / 1 urea pH 7.0, 0 ⁇ 0.2.
• Wash buffer 1 0.15 mol / 1 potassium phosphate, 6 mol / 1 urea pH 7.0 ⁇ 0.2.
• Wash buffer 2 0.02 mol / 1 potassium phosphate buffer pH 7.0 ⁇ 0.2.
• Elution buffer 0.5 mol / 1 potassium phosphate pH 7.0 + 0.2.
• Detection takes place at 254 nm with a UV detector / recorder unit.
• a 1% product solution (plasmid DNA), measured with a calibrated photometer, is used as the calibration solution.
• the Q-Sepharose pool is brought to a final concentration of 1.1 mmol / 1 calcium chloride and drawn onto the equilibrated column.
• the peak is collected and concentrated to approximately 50 ml with cross-flow filtration.
• the CFF is operated with a retentate flow rate of 100-200 l / h «m 2 , a transmembrane pressure of approx. 0.8 bar and an overflow pressure of approx. 1.2 bar carried out.
• the retentate is then fluidly diafiltered against TE buffer (10 mmol / 1 Tris / HCl, 1 mmol / 1 EDTA, pH 8.0) until the values for pH and conductivity of the retentate and TE buffer match. After the diafiltration process has ended, the retentate is adjusted to a plasmid DNA concentration of 1 mg / ml by dilution with diafiltration buffer.
• the intactness of the plasmid DNA obtained is checked by agarose gel electrophoresis.
• an aliquot of the plasmid DNA in various concentrations is applied to an agarose gel.
• the agarose gel shown shows lanes 1 and 10 of DNA length standard No. II (fragment sizes: 125, 564, 2027, 2322, 4361, 6557, 9416, 23130 bp) and lanes 2 and 9 of DNA length standard III (fragment sizes : 125, 564, 831, 947, 1375, 1584, 1904, 2027, 3530, 4268, 4973, 5148, 21226 bp).
• Lane 3 shows pBR322 (4162 bp) as a reference plasmid, which was purified using the conventional cesium chloride gradient method.
• plasmid DNA purified by this method essentially contains plasmid DNA which corresponds to the conformation "covalently closed circle” (dominant "supercoiled band”).
• plasmid DNA pCMV-CAT
• This plasmid DNA had been further purified following the process according to the invention via Q-Sepharose and hydroxylapatite chromatography, as well as cross-flow filtration.
• Lane 1 DNA length standard II (Boehringer Mannheim GmbH, cat.no.236250)
• Lane 2 DNA length standard III (Boehringer Mannheim GmbH, cat.no.528552)
• Lane 3 pBR322 (Boehringer Mannheim GmbH, Cat.No. 481238)
• Lane 9 DNA length standard III (Boehringer Mannheim GmbH, cat.no.528552) Lane 10 DNA length standard II (Boehringer Mannheim GmbH, cat.no.236250)
• the plasmid DNA purified according to the invention essentially shows a dominant band. This shows that the plasmid DNA obtained according to the invention is not damaged and remains in its original conformation. In addition, the lack of additional bands in the agarose gel shows that the chromosomal DNA contained in the digested cell suspension is not fragmented during the continuous centrifugation, but can be completely separated from the plasmid DNA as a precipitated macromolecule.

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• 1998
  • 1998-01-09 KR KR1019997006157A patent/KR100337046B1/ko not_active IP Right Cessation
  • 1998-01-09 TR TR1999/01606T patent/TR199901606T2/xx unknown
  • 1998-01-09 BR BR9807061-4A patent/BR9807061A/pt not_active IP Right Cessation
  • 1998-01-09 US US09/341,367 patent/US20020015982A1/en not_active Abandoned
  • 1998-01-09 WO PCT/EP1998/000104 patent/WO1998030686A2/de not_active Application Discontinuation
  • 1998-01-09 CA CA002277468A patent/CA2277468C/en not_active Expired - Fee Related
  • 1998-01-09 AU AU58627/98A patent/AU720911B2/en not_active Ceased
  • 1998-01-09 EP EP98901955A patent/EP0973883A2/de not_active Withdrawn
  • 1998-01-09 CN CNB988017636A patent/CN1142273C/zh not_active Expired - Fee Related
  • 1998-01-09 JP JP53055598A patent/JP2001512963A/ja active Pending

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