Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Lysis of micro-organism
Reexamination Certificate
1997-06-19
2001-08-14
Ware, Deborah K. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Lysis of micro-organism
C435S306100, C435S975000
Reexamination Certificate
active
06274371
ABSTRACT:
This invention is directed to a process for the preparation of nucleic acids from natural sources, such as
E. coli
cells, human and animal cells, by separating the lysed natural sources, such as cells or cell debris, in a sample by filtration, and to a kit containing means for performing said process.
Frequently, in the preparation of cell components, particularly nucleic acids, the problem arises to separate the lysed natural sources, from which the components are derived, from dissolved material. Usually, the separation of cells or cell debris is effected by centrifugation, whereby cell fragments or cells deposit as a pellet in the centrifuge tube. The dissolved cell components then are found in the supernatant and may be pipetted. In the preparation of nucleic acids, simple filtration methods were not successful for the separation of the lysed cells or their fragments because the cell fragments either pass through the filter having too large a pore size and thus give rise to turbidity and impurities in the filtrate or, when filters with appropriately narrow pores are used, inevitable jamming will result, so that a purposeful preparation of the cell components is no longer possible.
Thus, the present invention is based on the problem of providing a process and creating a device by means of which centrifugation steps for the preparation of cell components from natural sources, such as cells, may be avoided by using filtration steps which are easier to handle.
Conventionally, in order to isolate components from cells, the latter are lysed first. In the preparation of nucleic acids, the cells have to be lysed first, for instance, by using enzymes, such as e.g. proteinase K and lysozyme, detergents, such as SDS, Brij, Triton X 100, Tween 10, and DOC, and chemicals, such as sodium hydroxide, guanidine hydrochloride and guanidine isothiocyanate. The cell fragments are sedimented by centrifugation, and the supernatant is decanted or pipetted off and subsequently purified by chromatography or extraction with phenol or chloroform and an alcohol precipitation. Maniatis; Current Protocols in Molecular Biology, Ausubel, F. M. et al., eds. (1991), Wiley Interscience, New York; Birnboim H. C. and Doly, J. (1979), A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA. Nucl. Acids Res. 7, pp 1513-1522.
Such centrifugation in a conventional laboratory centrifuge, e.g., Heraeus Biofuge GL, Beckmann GS6, will take from 15 min to 2 h at from 3,000 rpm to 20,000 rpm, depending on the particular application and the particle size of the cell fragments. This means that with large sample numbers, the centrifugation will require much personnel and involve losses in time. Therefore, it is desirable to be provided with a simple and rapid process for the removal of the cell fragments.
In preliminary experiments, the filter materials and filtration methods which have been available to date have proven to be unsuited for the separation of such biological cell debris. Sterile filters, e.g. made of nylon or cellulose acetate, having pore sizes of from 0.2 &mgr;m or 0.45 &mgr;m will instantly clog and lack the capacity of retaining a larger amount of cell fragments.
These filters are only useful for the filtration of liquids having a very low content of solids or cellular contaminations.
In the filtration process according to the invention for the preparation of nucleic acid from natural sources, the sources containing nucleic acids are first lysed. The lysate is left to rest for some time. Preferably, the resting time is at least 1 minute, from 5 to 10 minutes being particularly preferred. The resulting lysate then passes a filter layer of glass, silica gel, titanium oxide, aluminium oxide or packed diatomaceous earth, or interwoven or cemented non-wovens of glass fibers and silica gel, as well as cellulose, paper, compressed paper, paper non-wovens. Thereafter, the fraction leaving the filter layer is collected and the nucleic acid is subsequently isolated and purified from the collected fraction.
Especially with a packed filter layer of silica gel having a particle size of from 15 to 30 &mgr;m, cell fragments can be retained successfully and without any clogging of the filter, and a clear lysate can be obtained.
Preferably, the filter layers are modified such that there is no affinity for nucleic acids. In particular, minerals bearing hydroxy groups or coated materials, such as diol-silica gel, diol-diatomaceous earth, and/or diol-perlite, may be used.
In a preferred embodiment, sample flow through the filter layer may be facilitated by applying positive or negative pressure. However, due to the pore size configuration of the filter layer, passage of the sample to be filtrated through the filter layer is also possible driven by gravity. Furthermore, in order to accelerate the passage of sample through the filter layer, the sample may also be passed through the filter layer by centrifugation.
As the filter layers, there are used, e.g., silica gel, glass or diatomaceous earth having particle sizes of from 5 &mgr;m to 100 &mgr;m.
A particularly preferred filter layer is untreated diatomaceous earth or modified diol-diatomaceous earth having flow values of from 0.1 to 15 Darcy.
The sources containing nucleic acids may be cells from cell cultures, any kind of tissues, body fluids or microorganisms.
The process according to the invention is particularly suitable for the preparation of plasmid DNA from
E. coli
, yeast or eukaryontic cells, or of genomic DNA from blood or cells, the DNA having a size of from 1 to 50 kb. The process according to the invention is also useful for purifying plasmid DNA, cosmid DNA, in particular for molecular-biological research, such as cloning and sequencing, plasmid DNA for gene therapy, genomic DNA for analytics, diagnostics and gene therapy, and/or viral nucleic acids.
The positive pressure at the side before the passage through the filter layer is preferably achieved by means of a piston.
Preferably, the filtration is followed by further processing steps, such as separation on anion exchangers and/or adsorption to and desorption from other mineral supports.
A device for performing the process consists of a hollow body in which the filter layer is arranged. The filter layer is preferably arranged between two fixing means. As said hollow body, there may be used a syringe, in particular.
As the filter layer which may be used in the process according to the invention, there may be used layers in the form of packings, in particular of glass, silica gel, titanium oxide, alumina, or diatomaceous earth, e.g. cellite or silica gel or perlite, but also interwoven or cemented non-wovens of glass fibers and silica, as well as paper, compressed paper, paper non-wovens, or combinations thereof. Other particles suitable for filtration made of minerals or synthetic polymers, diatomaceous earth, silica gel, perlite, and other mineral supports are either untreated or treated to have a hydrophilic surface which is hardly capable of adsorbing nucleic acids, e.g. diol modified diatomaceous earth.
In a preferred embodiment of the process according to the invention, multiple samples are processed simultaneously and passed through appropriate devices advantageously adapted to microtitration systems.
A suitable device for performing the process according to the invention consists of a preferably cylindrical hollow body having an inlet and outlet and a filtration means arranged in the hollow body. For securing the filtration means, common securing means may be used, such as, for example, cementings, but also securing by frictional forces by jamming the filtration means in the hollow body.
BRIEF DESCRIPTION OF THE DRAWING
The device consists of at least one filter layer with equal pore sizes as seen in the direction of outlet
60
.
FIG. 1
shows a particularly preferred variant of the device, where in the preferably cylindrical hollow body
40
, the filtration means
70
is designed from one sheet. The particle size of the filter layer is in the range of from 5 &mgr;m to 500 &m
Jacobson & Holman PLLC
Qiagen GmbH
Ware Deborah K.
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