Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-09
2001-06-05
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100
Reexamination Certificate
active
06242220
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology and particularly to the field of DNA isolation and purification. The present invention involves a method for producing covalently closed circular DNA (ccc DNA) from a cell line or a unicellular organism, preferably a bacterium, essentially free of genomic DNA.
BACKGROUND OF THE INVENTION
The production of covalently closed circular DNA in pure form has become increasingly important in the past two decades. The development of DNA vaccines in the last few years has added to this demand. The purification protocol for ccc DNA requires its separation from the genomic DNA of the cultured cell or unicellular organism carrying the ccc DNA.
Standard protocols designed to simplify this process as far as possible have the problem that after the degradation step for genomic DNA the sample still comprises the degrading protein or fragments thereof. A partial answer to this problem is provided by such suppliers as Epicenter Technologies (Madison, Wisconsin, USA), which distributes an ATP-dependent DNAse under the tradename Plasmid-Safe™ for the separation of plasmid DNA (ccc DNA) from genomic DNA. The enzyme specifically cleaves linear DNA (such as genomic DNA) while leaving ccc DNA intact. The earliest identification of an enzyme of this class, namely of the ATP-dependent exonuclease RecBCD, was published in 1972 (Goldmark and Linn,
J. Biol. Chem.,
247, 1849-1860).
In spite of such advances, proteinaceous impurities contained in the ccc DNA sample solutions generate problems when the ccc DNA is used for subcloning purposes. In addition, such impurities may cause adverse side effects when used in in vivo applications.
Thus, the technical problem underlying the present invention was to improve the existing protocols for separating ccc DNA from genomic DNA. In accordance with the present invention it was found that a novel process comprising a sequence of steps discussed in more detail below not only provides ccc DNA of a high purity grade but also removes proteinaceous impurities that could interfere with subsequent cloning steps or in vivo applications.
SUMMARY OF THE INVENTION
The method of the invention comprises the steps of precipitating a cleared lysate with an alcohol such as isopropanol and, after washing and resuspension of the precipitate, digesting the genomic DNA with a nuclease that cleaves linear DNA or circular DNA that includes a nick or comprises a free 3′ end or 5′ end but not ccc DNA. Finally, purified ccc DNA is separated from the remainder of the product of the digestion step by contacting said product with an ion exchange material. Preferably, the separation step is a chromatography step using an anion exchange resin, most preferably an anion exchange column. The invention also relates to purified ccc DNA obtained by the method of the invention, pharmaceutical compositions comprising said ccc DNA, and kits for carrying out the method of the invention.
Accordingly, the present invention relates to a method of producing covalently closed circular (ccc) DNA from a cell or unicellular organism wherein said ccc DNA is essentially free of genomic DNA comprising the steps of:
(a) precipitation of a cleared lysate of said cell or unicellular organism with 0.6-5 volumes of an alcohol;
(b) washing of the precipitate with an alcoholic solution;
(c) resuspension of the precipitate;
(d) digestion of the genomic DNA with a nuclease that cleaves linear DNA and/or circular DNA that comprises a nick or comprises a free 3′ or 5′ end but not ccc DNA; and
(e) separating purified ccc DNA from the remainder of the product of step (d) by contacting the product of step (d) with an ion exchange material, preferably an anion exchange material.
The present invention has the particular advantage that highly purified ccc DNA is obtained using an uncomplicated and comparatively inexpensive method. Yet, in spite of the method's simplicity, the critical sequence of steps can by no means be regarded as obvious. For example, when starting out to solve the aforementioned technical problems, attempts by the inventors to establish the step of employing a DNase directly after the lysis of bacteria by alkali treatment resulted in failure. This negative result is probably due to the presence of impurities, or salts, etc. in the lysate.
The method of the invention saves several hours of preparation time compared to the conventional procedures of adding the enzyme to a final stage of the DNA isolation process. Especially advantageous is the fact that with the current invention the use of toxic reagents like phenol or chloroform for the extraction of the remaining enzyme can be totally eliminated.
The ccc DNA obtained by the method of the invention can be used for a variety of purposes including cloning procedures and pharmaceutical applications such as vaccination. The ccc DNA may optionally be further purified, for example, by HPLC or may be prepared for storage, e.g., by lyophilization.
DEFINITIONS
The term “covalently closed circular DNA” as used herein refers to DNA molecules that have assumed a circular form in contrast to linear DNA molecules such as eukaryotic chromosomal DNA or bacterial chromosomal DNA that comprises a nick or comprises a free 3′ or 5′ end. Moreover, the circular structure of the above referenced DNA molecules is covalently closed. ccc DNA is well known in the art and is further described, for example, in K. G. Hardy (ed.),
Plasmid, a Practical Approach
(IRL Press Oxford U.K., Washington D.C., U.S.A., 1987).
The term “essentially free of genomic DNA” is intended to mean that more than 95% of the genomic DNA, preferably more than 98%, and most preferably more than 99% of the genomic DNA in a sample has been degraded and/or removed. Optimally, the ccc DNA isolated according to the invention is 100% purified or is as close to 100% purified as is within the limits of detection using standard assays.
The term “cleared lysate” is also well known in the art and refers to an aqueous solution containing plasmid DNA, RNA and proteins which is obtained after lysis of cultured cells or unicellular organisms and the separation of the cell debris, usually by filtration or centrifugation. For obtaining a cleared lysate, lysis of said cell or organism has to precede the steps recited above. Accordingly, in one embodiment, the method of the invention envisages an additional step that relates to the lysis of said cell or unicellular organism under conditions that leave the ccc DNA essentially intact. Methods for the lysis of cells and unicellular organisms are well know in the art and described, for example, in Sambrook et al.,
Molecular Cloning, A Laboratory Handbook,
2
nd
edition (CSH Press, Cold Spring Harbor, U.S.A. 1989).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to a method for isolating covalently closed circular (ccc) DNA essentially free of genomic DNA from a cell or unicellular organism. ccc DNA obtained following the method of the invention is highly pure and homogenous, typically containing no detectable genomic DNA from the source cells or microorganisms.
The method according to the present invention for producing covalently closed circular (ccc) DNA from a cell or unicellular organism, wherein said ccc DNA is essentially free of genomic DNA, comprises the steps of:
(a) precipitation of a cleared lysate of said cell or unicellular organism with 0.6-5 volumes of an alcohol;
(b) washing of the precipitate with an alcoholic solution;
(c) resuspension of the precipitate;
(d) digestion of the genomic DNA with a nuclease that cleaves linear DNA and/or circular DNA that comprises a nick or comprises a free 3′ or 5′ end but not ccc DNA; and
(e) separating purified ccc DNA from the remainder of the product of step (d) by contacting the product of step (d) with an ion exchange material, preferably an anion exchange material.
The method may be applied to the isolation of ccc DNA from any cell, whether prokaryotic or eukary
Schorr Jaochim
Wahle Stephan
Weber Martin
Berka Thomas R.
Brusca John S.
Kim Young
Qiagen GmbH
Yankwich Leon R.
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