Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2000-03-02
2001-07-10
Ketter, James (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S350000, C536S023100, C241S180000
Reexamination Certificate
active
06258930
ABSTRACT:
SPECIFICATION
The analysis of biological materials, particularly using nucleic acid-based methods, is becoming increasingly important in many fields, particularly in basic biological and medical research, and especially in applications such as molecular diagnostics or animal husbandry and horticulture.
In order to be able to analyse biological materials first of all the biological sample material has to be broken down. First of all, the cell structure has to be destroyed in order to allow biological molecules to be isolated. Starting materials such as plants, bacteria or yeasts, moreover, have a highly resistant cell wall which makes it even more difficult to release the molecules which are to be analysed, particularly nucleic acids and/or proteins.
A decomposition process which is suitable for breaking down these materials should ensure that the samples can be broken down very rapidly, if possible within a few seconds, and at the same time in a highly efficient manner. This is particularly important when isolating nucleic acids, particularly RNA, since many gene expression analysis methods require quantitative isolation of intact nucleic acid, particularly RNA. RNA is a material which degrades very rapidly after the destruction of the cell structure and is therefore impossible to analyse when unsuitable slow methods of breaking down are used.
Numerous applications of nucleic acid-based analysis such as tissue typing, screening of new active substances for pharmaceuticals or quality control of seeds are characterised by a high throughput of samples and the use of small amounts of starting material. For many high throughput applications, the breaking down of the biological sample is the limiting step, since there are already automated systems available for further processing the sample once it has been broken down, e.g. the BioRobot made by QIAGEN.
A number of mechanical breaking down processes are described in the prior art. One such apparatus is described in the company brochure of the applicants Retsch “Vibration Mill (“Schwingmühle”) MM 2000”; insofar as it discusses the breaking up of biological cells, holding means designed to hold five or ten single-use test tubes can be clamped in the holder of the vibrating ball mill, the test tubes being inserted into the holding means for the break down process and at least one glass bead being placed into each individual test tube in order to break down the biological material. The mount provided on the vibrating ball mill for clamping the holding device in place or, if the vibrating ball mill is used for other grinding purposes, for clamping suitable grinding cups, consists of a U-shaped frame with a baseplate attached to the drive of the vibrating ball mill and with two opposing, planar retaining arms which hold the holding device or grinding cup between them, one of which is movably mounted and can be brought into clamping abutment on the holding device or grinding cup by means of an adjustment and clamping screw.
This known apparatus has the disadvantage that the break down of biological materials is not reproducible in different batches and therefore cannot be evaluated with sufficient accuracy, as the holding device in question cannot be clamped in exactly the same position between the holding arms of the mount and may change its position, however slightly, even during a breaking down process, depending on the tension applied by means of the adjustment and clamping screw; however, the position of the spindle of the holding device is not thereby fixed in spatial relationship with the drive of the vibrating ball mill. However, since the amplitude of the oscillating movement acting on the holding device during the break down adjusts itself as a function of the spatial relationship of the axis of the holding device to the drive, different amounts of energy are put into the grinding means with different degrees of tension or with a varying tension, which means that the biological material is broken down to different extents. A further disadvantage is that the break down capacity, for a batch of five or ten test tubes, is too little, by design.
A high sample throughput is particularly important for use in nucleic acid-based analysis methods and diagnostic processes. In addition, the break down process should avoid cross-contamination.
Hitherto, the prior art has not described any break down processes which would enable more than ten samples to be subjected to simultaneous, rapid, efficient breaking up as free from cross-contamination as possible.
The aim of the present invention is therefore to provide a rapid and effective mechanical method of breaking down, including an apparatus suitable for this purpose, which allows more than ten samples to be subjected to simultaneous, rapid and efficient breaking down as free from cross-contamination as possible.
This problem is solved according to the invention by the use of multi-well blocks containing more than ten wells for breaking down biological samples in a vibrating ball mill, and by the process recited in claim
2
, as described in more detail hereinafter, and by the apparatus described in claim
9
. Preferred embodiments of the process according to the invention and the apparatus according to the invention are recited in the subclaims.
Specifically, the invention relates to use of multi-well blocks having more than 10 wells for breaking down biological samples in a vibrating ball mill.
The terms “biological material” or “biological sample” for the purposes of the present invention denote all materials which are produced by biological organisms or can be isolated from them; in particular, they denote materials which contain nucleic acids and/or proteins. The term “biological material” or “biological sample” includes untreated or pretreated samples, e.g. plasma, body fluids, particularly blood, sputum, urine, faeces, sperm, cells or cell cultures, serum, leukocyte fractions, smears, tissue samples of all kinds, plants and parts of plants, microorganisms such as bacteria, viruses such as cytomegalo virus, HIV, hepatitis B, hepatitis C, hepatitis &dgr; virus, yeasts, embryos, fungi, cell-free sample material, etc. The term “biological sample” also includes both a mixture of the abovementioned samples such as fungus-infected plants or whole human blood containing mycobacteria as well as food samples which contain free or bound nucleic acids or cells containing nucleic acids, environmental samples which contain free or bound nucleic acids or cells containing nucleic acids. Pretreated biological samples may be, for example, heat treated —frozen, dried, etc., or chemically treated, e.g. fixed in suitable chemicals such as formalin, alcohol, etc.
Biological molecules for the purposes of the present invention are organic molecules or derivatives thereof which are part of living cells or organisms, particularly nucleic acids and/or proteins, particularly DNA and/or RNA.
The term nucleic acids for the purposes of the present invention includes all possible kinds of nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid of all lengths and configurations, such as double-stranded, single-stranded, circular and linear, branched, etc., plasmids, viral and bacterial DNA and RNA as well as genomic or other non-genomic DNA and RNA from animal and plant cells or other eukaryotes, t-RNA, mRNA in processed and unprocessed form, hn-RNA, rRNA and cDNA and all other conceivable nucleic acids. In addition, within the scope of the present invention, the definition includes a sample which contains nucleic acid or a sample or mixture of samples which contains nucleic acid, which may be used as suitable educts for “downstream applications” such as in vitro transcription, PCR reactions or cDNA syntheses.
The vibrating ball mill intended for using multi-well blocks is based on a vibrating ball mill having containers to be clamped thereon in a U-shaped holder having two opposing holding arms, one of the holding arms being movably mounted in order to clamp the grinding container and having an adjusting
Anghel Radu
Bastian Helge
Gauch Simone
Orth Uwe
Roord Manfred
Berka Thomas R.
Ketter James
Qiagen GmbH
Yankwich Leon R.
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