Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Patent
1993-02-23
1994-10-04
Warden, Robert J.
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
21032164, 21032171, 21032175, 435259, 435 6, 436178, 536 254, 536 2541, B01D 6300, C12Q 168, C12N 106, C12N 108
Patent
active
053526090
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a cartridge for extracting and purifying nucleic acids, in particular genome DNA, from a sample of blood, of tissue cells, or of culture cells, and also to apparatus and a method making use of said cartridge.
Conventional methods for extracting DNA from cells generally consist in total lysis of the cells, in degradation of the proteins by means of enzymes, in removing the degraded proteins and the lipids by means of phenol and chloroform, in precipitating the DNA by means of ethanol, and then in putting the extracted DNA back into suspension.
To extract DNA from the blood of mammals, the treatment comprises lysis of the red corpuscles and of the plasma membranes of the white corpuscles, leaving the nuclei of the white corpuscles intact, and then separating out said nuclei by centrifuging and subsequently treating them as described above.
With solid tissue, the tissue is broken up and homogenized in an appropriate solution before being subjected to the above-described treatment.
The above treatments are lengthy and fiddly and they lend themselves poorly to extracting genome DNA from a large number of cell samples. In addition they require toxic solvents to be handled such as phenol and chloroform.
Simpler methods have already been proposed (see for example "Nucleic Acids Research" Vol. 17, No. 20, 1989, page 8390) essentially consisting in cell lysis, in isolating the nuclei by centrifuging, in nucleus lysis, and in degrading the proteins with a mixture of PLB and of K proteinase, while avoiding the use of solvents such as phenol and chloroform.
An essential object of the present invention is to provide a cartridge for extracting and purifying nucleic acids such as genome DNA from a cell sample, enabling the extraction of DNA to be further simplified by eliminating the centrifuging separation operations used in the prior art, and also eliminating extraction by means of organic solvents, precipitation of the DNA, and putting it back into suspension (a step which is often very time consuming).
Another object of the invention is to provide apparatus and a method for extracting and purifying nucleic acids such as genome DNA, making use of said cartridge.
To this end, the invention provides a cartridge comprising a dialysis enclosure in particular for purifying nucleic acids, and inlet and outlet means for admitting and extracting substances into and out from the dialysis enclosure, the cartridge being characterized in that it comprises a thin elongate plane support having a central slot, with dialysis membranes being fixed on two faces thereof with the dialysis enclosure being defined between them, and two end rings which are mounted on longitudinally opposite ends of said support, each including a through duct that opens out into the dialysis enclosure.
Such a cartridge can be made having dimensions that enable it to be mounted in a standard tube of a conventional device for treating biological samples, e.g. a carousel type device as is widely used in biological laboratories.
Advantageously, the cartridge of the invention comprises a filter for retaining the cell nuclei and carried by the support to separate the dialysis enclosure into two separate compartments into which the through ducts of said rings open out on respective opposite sides of the filter.
The above-mentioned filter may, for example, be a polyester film having very fine mesh size with dimensions of the order of 1 micrometer.
The presence of this filter in the dialysis enclosure of the cartridge makes it possible to avoid the conventional operations of separating out the nuclei by centrifuging, because the cell nuclei are retained and fixed selectively on the filter when the lysed cells are caused to flow in the dialysis enclosure through the filter.
In a preferred embodiment of the invention, the above-mentioned support is made up of two superposed rectangular frames, each frame having a substantially axial groove on the inside-face of one of its short sides, which groove has one end opening out into the dialysis enclos
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Bertin & Cie
Carpenter Robert
Warden Robert J.
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