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

Definitions

• the present invention relates to a method for the rough clarification of cell disruption of microorganisms, in particular bacteria - such as E.coli.
• plasmids are generally gram-positive or gram-negative bacteria - such as E. coli mutants.
• a particular problem in this connection is the removal of unwanted genomic DNA, RNA and endotoxins that originate from the microorganisms used for the multiplication. Since the nucleic acids mentioned belong to the same class of biomolecules, they also have certain comparable physicochemical properties, such as plasmid DNA, and therefore represent an enormous difficulty in the purification or separation of the plasmid DNA.
• the cell disruption of microorganisms represents the initial step in the isolation or purification of plasmid DNA from such microorganisms.
• a lysis buffer such as SDS (sodium dodecyl sulfate) in the presence of sodium hydroxide - there are adjacent the fragments of the cell wall in the reaction solution to which the genomic DNA is bound via membrane-associated proteins.
• SDS sodium dodecyl sulfate
• this bond is rather weak, so that mechanical influences are sufficient to break these bonds.
• the genomic DNA released in this way into the aqueous supernatant is then not more easily separated from the plasmid DNA or RNA in later purification steps.
• the formation of flocculent precipitates occurs during the work-up process after neutralization or acidification of the solution resulting from the above-mentioned basic lysis, for example by means of an aqueous potassium acteate solution.
• These precipitates consist of potassium dodecyl sulfate (KDS, the potassium salt of SDS) which precipitates together with the cell wall and the cell membranes and other cell components due to attachment and denaturation to or from cell wall-associated proteins.
• KDS potassium dodecyl sulfate
• SDS potassium salt of SDS
• the genomic DNA anchored to the cell wall is thus also bound in these complexes.
• the KDS-protein-cell wall complex can be separated off by centrifugation, which at the same time also largely removes the genomic DNA.
• Centrifugation increased the proportion of genomic DNA released from the KDS precipitate in the aqueous supernatant.
• Filtration results in blocking and shear problems on a preparative scale while the filter is loaded with flake-contaminated bacterial lysates. This would also contaminate the plasmid phase with the genomic DNA in the filtrate. Another disadvantage is that the filter is often clogged.
• the object of the present invention is therefore primarily to provide a method for obtaining plasmid DNA or RNA which is preparative
• Scale is applicable and in which the plasmid DNA or RNA can be obtained largely free of genomic DNA and endotoxins.
• Another object of the present invention is to provide a method which is as simple and practical as possible and which avoids the disadvantages known from the prior art.
• the above-mentioned tasks are solved in that a cell disruption of biological material (microorganisms) is produced in a manner known per se from the prior art.
• the resulting reaction mixture is subjected to a change in pressure, ie an increase or decrease in pressure compared to normal pressure, with a decrease in pressure being preferred.
• the surprising advantage of this process step is that by changing the pressure of the atmosphere surrounding the reaction mixture according to the invention, the flakes float or sink, which ultimately results in a spatially highly compressed flocculent phase.
• the flaky phase settles on the liquid phase and can be separated very well and completely from the liquid phase.
• the pressure of the atmosphere surrounding the reaction mixture is preferably set to a value which is 200 to 1000 mbar lower than the normal pressure (atmospheric pressure under normal conditions). A reduction in pressure by a value in an interval of 300 to 800 mbar is particularly preferred.
• a similar effect occurs when a pressure is selected above the ambient pressure - preferably from 200 to 5000 mbar and particularly preferably from 200 to 2500 mbar - above the normal pressure. This measure also results in the formation of a flocculent phase which, however, does not float on the liquid in this case but sinks.
• phase separation leads to a spatially highly compressed flocculent phase, which increases the volume fraction of the product-containing aqueous phase at the same time.
• the process times for the clarification of cell disruption - such as significantly reduce in plasmid DNA production using E.coli, since the filtration units used do not block more or more slowly, and higher flow rates can be selected.
• the yield can be increased by about 10 to 30% with the method according to the invention (since the phase containing the flocculate is significantly more compressed than the corresponding phase, which can be produced under normal pressure conditions).
• the compacting of the flocculate which can be achieved according to the invention, is extremely low-shear, so that - as mentioned above - there is a risk of the genomic DNA and endotoxins being detached from the fragments of the cell wall
• Another advantage of the method according to the invention is that the filters block significantly more slowly in subsequent filtration steps than when an unclarified bacterial lysate is used.
• the starting material is subjected to an alkaline lysis known from the prior art.
• a vacuum is applied at a pressure which is 200 to 1000 mbar lower than the normal pressure (air pressure). A reduction in pressure by a value in an interval of 300 to 800 mbar is particularly preferred.
• an ambient pressure above normal pressure is selected - preferably in an interval of 200 to 5000 mbar and particularly preferably in an interval of 200 to 2500 mbar - above normal pressure.
• Fig. 1 shows a reaction mixture before using the method according to the invention (Fig. 1A) and after applying a vacuum (Fig. 1B) on a 50 ml scale.
• FIG. 2 shows a lysis mixture according to Example 3 on a 50 liter scale - as in FIG. 1 - before applying a vacuum (FIG. 2A) and after using the method according to the invention (FIG. 2B).
• 68 g of frozen pUC19 biomass are thawed in a beaker and in one liter of a commercially available resuspending buffer (P1 buffer from QIAGEN, D-40724 Hilden) and homogenized by shaking. Then 1 liter of a commercially available cell lysis buffer is added (P2 buffer from QIAGEN, D-40724 Hilden) and the biomass is subjected to the lysis. The lysis process is e.g. supported by shaking. The incubation period for this step is at least 5 minutes. The lysate obtained in this way is transferred to a suitably large reaction vessel for clarification (volume approx.
• Resuspending buffers (P1 buffer from QIAGEN, D-40724 Hilden) thawed and resuspended.
• 15 ml of a commercially available cell lysis buffer (P2 buffer from QIAGEN, D-40724 Hilden) are then added, and the biomass is subjected to lysis and incubated on ice for a period of about 5 minutes.
• the resulting lysate is mixed with 15 ml of an ice-cooled commercial neutralization buffer (P3 buffer from QIAGEN, D-40724 Hilden) and shaken.
• the reaction vessel is transferred to a desiccator and exposed to a vacuum of 0.7 bar for about 7 minutes. An immediate floating of the solid components can also be observed.
• the cell lysates (10 x 4.5 liters) thus produced are carefully decanted into a 50 liter clarifying bottle.
• the bottle internal pressure is then reduced to an ambient pressure of 600 mbar (corresponds to -400 mbar compared to normal pressure).
• the vacuum is maintained until all flakes float on the surface. The process takes about 5 - 10 minutes.
• the vacuum is then released and the clear cell lysate is drained from the bottom of the clarifying bottle until the flocculating KDS phase reaches the lower outlet tap.
• the liquid phase thus obtained is free from visible flakes according to visual inspection.
• 1 liter LB Miller is inoculated with the E.coli DH5 alpha RCB strain and incubated in a shaker for approx. 12 h at a temperature of 37 ° C.
• the bacteria are then centrifuged off.
• the pellet obtained in this way is resuspended in 75 ml of a commercially available resuspension buffer without RNase (for example P1 buffer from QIAGEN, D-40724 Hilden) and added with 75 ml of a commercially available lysis buffer (for example P2 buffer from QIAGEN, D-40724 Hilden) mixed by gentle shaking.
• a commercially available neutralization buffer e.g.
• the subsequent cleaning e.g. column chromatographic purification via the steps: binding the RNA to the matrix, washing the bound RNA with a suitable washing buffer and subsequent elution with subsequent precipitation of the RNA from the eluate with isopropanol, provides the RNA in a total yield of 720 ⁇ g of good quality RNA.

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• 2002
  • 2002-02-20 DE DE10207170A patent/DE10207170A1/de not_active Withdrawn
• 2003
  • 2003-02-20 US US10/504,258 patent/US7214508B2/en not_active Expired - Lifetime
  • 2003-02-20 EP EP03706546A patent/EP1478749B1/de not_active Expired - Lifetime
  • 2003-02-20 WO PCT/EP2003/001769 patent/WO2003070942A1/de not_active Ceased
  • 2003-02-20 AT AT03706546T patent/ATE464380T1/de active
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