Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-11-18
2004-11-23
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S025300, C536S025400, C536S025410
Reexamination Certificate
active
06821757
ABSTRACT:
The invention is concerned with a method for separating circular nucleic acids from a mixture having different species of nucleic acids. Furthermore, an aqueous buffer for the method is disclosed.
Many techniques in molecular biology require pure nucleic acids in general and plasmid DNA in particular.
Plasmids are double stranded circular closed DNA molecules which are found in certain organism additional to chromosomal DNA. Examples for such organism are some yeast and plant cells and all species of bacteria. While chromosomal DNA comprises all the information necessary for the cells to live, natural occurring plasmids are an additional genetic element providing and advantage to the host cell under certain environmental conditions.
Plasmids are a preferred tool in molecular biology due to the fact that they replicate autonomously from the chromosomal DNA and that they—in other than chromosomal DNA—can be isolated from bacteria cells in intact form. They can be hydrolysed at specific recognition sequences with commercially available restriction endonucleases, joined with fragment of foreign DNA, amplified with DNA polymerases and transferred into suitable cells.
The isolation of plasmid DNA is therefore often a prerequisite for subsequent molecular biological experiments, such as PCR reactions, sequencing reactions, cloning reactions, restriction hydrolyses, transformations and transfections.
Several methods for the isolation of plasmid DNA from bacterial cells are known. Common to all these methods is that they follow the scheme:
1. Formation of cleared lysate and
2. purification of plasmid DNA from the cleared lysate.
The steps for the formation of cleared lysate are almost identical between the different methods, characteristic differences only occur during the purification of plasmid DNA from the cleared lysate.
The formation of cleared lysate comprises the steps of:
cell lysis
precipitation of cellular components and subsequent
removal of the precipitate from the plasmid containing solution to form cleared lysate.
Cell lysis is usually realized under alkaline conditions in the presence of sodium dodecylsulfate resulting in bacterial crude lysate. To precipitate cellular components like chromosomal DNA, proteins, cellular debris etc., a potassium or sodium acetate buffer is added to the crude lysate, which adjusts the mixture to slightly acidic pH (4.8 to 5.0). Plasmid DNA does not precipitate under these conditions, so that it remains in the supernatant. In order to form a cleared lysate, the precipitate is removed from the plasmid containing solution either by centrifugation (Sambrook J., Fritsch E. F. and Maniatis T, (1989), “Molecular Cloning. A Laboratory Manual”, pp. 7.49 to 7.50, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Maniatis, Laboratory Manual), filtration (EP 0 616 638 B1) or magnetic separation using magnetically attractable beads which do not specifically bind the precipitate (U.S. Pat. No. 5,681,946 and U.S. Pat. No. 5,523,231). M. A. Marko et al. discloses in “Analytical Biochemistry”, Vol. 121, No. 2, Apr. 19, 1982, pp. 382-387, a preparative procedure for obtaining highly purified plasmid DNA from bacterial cells. The method is adapted from an earlier procedure which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the method described all detectable RNA, chromosomal DNA and protein are removed without the use of enzymes phenol extraction dialysis or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 M sodium perchlorate is used for the final purification step.
WO 95/01359 discloses a chromatographic purification and separation process for mixtures of nucleic acids. The nucleic acids to be separated and purified are adsorbed on a substrate from a solution which has a high salt concentration (ionic strength) and/or a high alcohol concentration, then desorption of the substrate is carried out by means of a solution with low salt concentration (ionic strength). The process in characterized in that the mixture of nucleic acids is adsorbed on a porous or non-porous mineral substrate made of metal oxides and/or mixed metal oxides, silica gel, materials composed mainly of glass, aluminium oxide, zeolithes, titanium dioxide, zirconium dioxide. The mixture of nucleic acids is adsorbed on the substrate from an aqueous adsorption solution with a high salt concentration (ionic strength) and with 1 to 50% by volume aliphatic alcohol with a 1 to 5 carbon atoms long chain, and/or polyethylene glycols (PEG) and/or hydrophobic, inorganic and/or organic polymers and/or organic acids, such as trichloracetic acid (TCA). If required, the mixture of nucleic acids is then washed with a washing solution, eluted with a solution having a lower salt concentration (ionic strength) and the thus obtained nucleic acid or nucleic acid fraction is collected. Carter, M. J., and Milton, I. D., disclose in “Nucleic Acids Research, 1993, Vol. 21, No. 4, Jan. 11, 1993 an inexpensive and simple method for “DNA purifications on silica particles. The method is a dissolving process concerning an already highly purified nucleic acid fraction which is bound to a matrix under non alkaline conditions.
WO-A 97/29190 discloses a scalable method for the production of highly purified plasmid DNA in
E. coli
. The method includes growing plasmid-containing cells to a high biomass in exponential growth and lysing the cells by raising the pH of the culture to a carefully controlled pH value in which chromosomal DNA is denatured but plasmid DNA is reversibly renatured. The method has been developed for the production of pharmaceutical grade DNA for use in in vivo and ex vivo gene therapy.
The methods for the purification of plasmid DNA from cleared lysate can be summarized —according to their underlying principle—into different groups.
One of these make use of density gradient centrifugation. This technique separates the components of the cleared lysate, like residual genomic DNA, RNA, proteins etc., according to their size in a caesium chloride gradient. The fraction, containing the plasmid DNA, is sucked off the centrifugational tube, further purified from salts by dialysis and finally concentrated by ethanol precipitation.
Methods of another group are based on the principle liquid-liquid extraction. The cleared lysate is several time extracted with phenol or a mixture of phenol and chloroform or a mixture of phenol, chloroform and an alcohol. During these extraction steps proteins, chromosomal DNA and other residual cellular impurities are transferred into the organic phase, while leaving the plasmid DNA in the aqueous phase. Traces from phenol were extracted several times with chloroform or a mixture of chloroform and an alcohol. The DNA is finally purified and concentrated by ethanol precipitation.
Other methods are based on anion exchange chromatography. The cleared lysate is applied on anion-exchange resin under appropriate salt and pH conditions. The binding conditions are thus adjusted that plasmid DNA are bound to the anion-exchange material but not impurities like RNA, Proteins, residual genomic DNA. After washing out impurities under salt conditions adjusting medium ionic strength, pure plasmid DNA is eluted under high ionic strength salt conditions. To remove the salt and to concentrate the plasmid DNA a final ethanol precipitation is necessary.
Purification of plasmid DNA using silica material is another basic principle. It uses the fact that DNA adsorb to silica material in the presence of chaotropic substances. The cleared lysate is mixed with a chaotropic buffer and subsequently applied to silica material, either a silica membrane or loose silica particles. After removal of the salts by a washing step DNA is eluted with a low salt buffer or water.
All known methods for the isolation of plasmid DNA from bacteria comprise the formation of cleared lysate. All known methods for clearing the bacterial lysate after precipitation of cellular particles, centrifugation, filtration and mag
Kang Jie
Sauer Philippe
Jacobson & Holman PLLC
Prats Francisco
QIAGEN GmbH
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