Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-09-09
2003-07-01
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S091100
Reexamination Certificate
active
06586585
ABSTRACT:
The present invention pertains to a method for the size reduction of high-molecular structures according to claim
1
, a device according to claim
7
, and uses of the device according to claims
12
to
15
.
Many biological isolation methods proceed from cell and tissue lysates, for example, which are frequently highly viscous systems. Due to their high viscosities, such systems can hardly be further processed and are usually subjected to various processes for homogenization or viscosity reduction.
In particular in the isolation of mRNA, it is necessary to homogenize the samples containing these substances in order to obtain non-viscous lysates or solutions. The preparation of homogeneous cell lysates or solutions having low viscosities is necessary in a number of techniques of molecular biology. Some of these techniques will be mentioned in the following for illustrative purposes.
Direct isolation of whole RNA or mRNA from cell or tissue lysates in most cases requires a homogenization step since the hybridization rate between the oligo-dT matrix necessary for mRNA isolation (e.g., cellulose, paramagnetic particles or latex particles) is very low in highly viscous solutions, as opposed to homogeneous solutions. The corresponding matrices may quasi agglomerate with the high-molecular DNA which is also present in the sample and render purification so difficult as even to become impossible in some instances.
Finally, in the preparation of whole RNA, as described, e.g., in German Patent P 43 21 904, the membrane which is necessary for chromatographic purification and is inserted in an apparatus will soon be clotted by highly viscous lysates or solutions.
The protocols for direct mRNA isolations, as known in the prior art using known products, consider that homogenization of the samples containing the substances mentioned is inevitable.
For example, it has been shown that in the polymerase chain reaction (PCR) with genomic DNA better results are achieved if sheared, low molecular weight DNA is employed. This could be related to the fact that smaller DNA fragments are more easily denatured and thus hybridization of the primer (primer annealing) is more efficient. In this connection, it is important that in preparations of nucleic acids (mRNA, whole RNA, and DNA) for PCR, cross contaminations with nucleic acids between samples which are processed simultaneously or succesively and cross contaminations with nucleic acids from other sources are by all means to be avoided since PCR is extremely sensitive so that contaminations with undesired nucleic acids would also be amplified correspondingly.
In the prior art, the following methods are employed essentially for the homogenization or shearing of tissues, cells and/or solutions containing high-molecular substances.
The size reduction (shearing) of high-molecular DNA may be performed by mechanically comminuting a sample deepfrozen in liquid nitrogen by means of mortar and pestle. Also, DNA will be sheared by repeated drawing of the sample containing high-molecular DNA through a cannula into a syringe. In addition to their high expenditure of work, these methods are not capable of ensuring a neat operation. Thus, the homogenization process for the isolation of biomolecules from potentially infectious starting materials, such as human cell and/or tissue samples (biopsy material), is found to be the most dangerous source of contamination of the operator since the homogenizate can squirt over a considerable distance in the conventional homogenization method.
Extraction of nucleic acids and other biomolecules from plant materials is found to be extremely difficult due to the high content of polysaccharides, polyols and/or other secondary metabolites since the substances mentioned form highly viscous lysates or gel-like structures in the commonly employed solutions following lysis of the cells or tissues. Simple centrifugation of the gel-like mass is not successful in most cases since a separation from the solution employed cannot be achieved. The gel-like structures and the high viscosity prevent efficient isolation of nucleic acids or render such isolation altogether impossible in some cases.
For the shearing of high-molecular DNA, there may also be employed specific homogenizers, such as the commercial Ultraturrax, Polytron, Omni, Tissuemizer, and the like. Although this method enables simple and rapid homogenization of virtually any sample, it has a drawback in that specific minigenerators have to be acquired for the homogenization of very small cell, tissue or solution quantities, in particular those which are designated for subsequent analysis by PCR or RT-PCR. In order to avoid cross-contaminations between different cell or tissue samples or other samples in this case, miniaturized disposable generators may also be employed which requires, however, that a relatively expensive device for homogenization is already available. In addition, the miniaturized disposable generators are very costly by themselves.
Another method for homogenization which has proved successful for some applications is ultrasonic treatment of the corresponding sample. In the field mentioned, for example, in PCR applications, this requires expensive equipment. Another drawback is the fact that disposable ultrasonic heads are not available. In addition, there is a risk that the nucleic acids may be too much fragmented by ultrasonic treatment, perhaps until being entirely unsuited for further analysis methods.
DE 41 39 664 A1 pertains to a device and a method for the isolation and purification of nucleic acids. The device for performing the method described consists of a hollow body having an inlet and an outlet wherein a particulate first material based on silica gel is arranged within the hollow body between two fixation means and a second material is arranged between the first material and the outlet, the first and second materials having different adsorption characteristics for nucleic acids.
DE 40 34 036 pertains to a device and a method for the isolation of nucleic acids from cell suspensions. The device for performing this method possesses a cell-uptaking matrix within a hollow body between two porous means. The pore size of said means is larger than the void size of the material forming the matrix.
CLONTECH Labs 1993, “Nucleic Acid Purification with CHROMA SPIN Columns”, pertains to a method for the extraction of nucleic acids from agarose gel pieces.
In D. Blöcher and G. Iliakis, Int. J. Radiat. Biol., 1991, vol. 59, 919-926, DNA fragments. are removed from a filter in the course of a non-denaturing filter elution. This appears to involve degradation of DNA fragments at the filter membrane.
Thus, the object of the invention is to provide a method which avoids the drawbacks mentioned of the prior art. A method is to be provided by which efficient preparation of nucleic acids in a simple and cost-saving way without cross-contaminations and, in general, homogenization of viscous systems, can be achieved.
The object of the invention is achieved by a method with the features of claim
1
. The subsequent subclaims
2
to
6
pertain to preferred embodiments of the method according to the invention.
Claims
7
to
11
pertain to a device which is particularly suitable for performing the method according to the invention. Claims
12
to
15
pertain to the use of the device according to the invention.
The method according to the invention for the size reduction of high-molecular structures, in particular nucleic acids, starts with charging the system containing the structures to be size-reduced onto a means. Said means includes at least one porous layer which porous layer has an asymmetrical pore size distribution. The pore size decreases in the direction of the passage of the structures to be size-reduced through the porous layer. The term pore size is to be construed as meaning an average pore size. The system containing the high-molecular structures to be size-reduced passes said means, said high-molecular structures being size-reduced during such passage, presuma
Owens, Jr. Howard V.
Qiagen GmbH
Wilson James O.
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