Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Patent
1990-11-26
1994-07-19
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
435289, 536 254, 935 19, E12N 108, C12M 136, C07H 1512
Patent
active
053309146
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an apparatus and a method for automatic purification of extra-chromosomal DNA from a cell suspension in a container.
It is known from Birnboim and Doly (Nucl Acids Res 7, 1513-1523, 1979) that plasmid DNA can be purified from Escherichi coli. The bacterial cells are first lysed in an alkaline medium containing a detergent. This is followed by a neutralizing step which yields the plasmid DNA in soluble form while most proteins, cell debris and chromosomal DNA precipitate. Plasmid DNA is then recovered from the mixture by centrifugation and a pure plasmid fraction is obtained by subsequent purification. Kieser (Plasmid 12, 19-36, 1984) has described a similar method, involving a combination of heat treatment and alkali treatment, which gave improved purification of plasmid DNA and less contaminating RNA. In these known methods, the purification involves several separate steps, such as for instance extractions and centrifugations, thus requiring considerable investments in equipment and operator time.
An automated plasmid purification work station based on a laboratory robot has been described by de Bonville and Riedel (Advances in Laboratory Automation Robotics, 1986, ed. Strimatis and Hawk, Zymark Corp., Hopkinton, Ma., pp353-360). That system is based on a number of laboratory unit operations and the robot uses several hardware stations, including a centrifuge, pipettes, capping devices, sample tubes, tip racks, microtiterplate etc.
Although this workstation is automated the system has several disadvantages. Firstly the system has to be set up with tip racks, sample tubes, supernatant tubes etc., which is labour intensive. Secondly, a considerable amount of disposable material is used which increases the expenses for each plasmid purified by the system. Thirdly, several mechanical operations are involved and this might cause technical problems in routine use.
An automated nucleic acid extractor has been described (EP-O 245 945) for extracting and purifying nucleic acids from cells without the use of centrifugation. In the method (described in EP-O 215 533) a lysate is created and mixed with a phenol-based solvent system and then heated to promote phase separation. The lower organic phase is removed and the upper aqueous phase is repeatedly extracted. The aqueous phase is finally dialyzed to further purify the nucleic acid solution. The method is used for extraction of high molecular weight DNA (about 10.sup.8 daltons) from eukaryotic cells such as peripheral blood lymphocytes and liver cells. Thus, the method is well suited for extraction of chromosomal DNA from cells, but is not adapted for purification of extrachromosomal DNA from bacteria, such as plasmid DNA, as the unit operations used in the method can not efficiently separate chromosomal from extra-chromosomal DNA.
The object of the invention is to bring about an apparatus and a method for purification of extrachromosomal DNA from bacteria that do not have these disadvantages.
This is attained in that the apparatus according to the invention for automatic purification of extra-chromosomal DNA from a cell suspension in a container, comprises chamber, therein, precipitate chromosomal DNA and, optionally, proteins and cell debris therein, collecting device, collecting device, the precipitate remaining therein, and
The method according to the invention for automatic purification of extra-chromosomal DNA from a cell suspension, comprises contents thereof to lyse the cells therein, the contents thereof to precipitate chromosomal DNA and, optionally, proteins and cell debris therein, liquid contents to a collecting device, contents thereof to dissolve the precipitate remaining therein, and
BRIEF DESCRIPTION OF THE DRAWINGS
The apparatus and method according to the invention will be described more in detail below with reference to the accompanying drawings on which
FIG. 1 shows a schematic block diagram of an embodiment of an apparatus according to the invention
In the block diagram (FIG. 1), 1 denotes a mixing chamber around whi
REFERENCES:
patent: 5057426 (1991-10-01), Henco et al.
Just et al. "Rapid High-Yield Purification of Plasmids by Alkaline Extraction . . . " BioTechniques vol. 1 No. 3 (1983) pp. 136-140.
Micard et al "Purification of RNA-Free Plasmid DNA . . . " Analytical Biochemistry vol. 148 (1985) pp. 121-126.
Chemical Abstracts, 103, 1985, 67838t (Micard et al.).
Chemical Abstracts, 103, 1985, 192722b (Ranhand et al.).
Chemical Abstracts, 100, 1984, 31842P (Just et al.).
Moks Tomas
Uhlen Mathias
CEMU Bioteknik AB
Reardon Timothy J.
Wityshyn Michael G.
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