Many biological substances, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances e.g. after lysis of cells. This makes them difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific analytes, e.g. nucleic acids, or specific analyte properties and play a major role in the field of diagnostics and bioanalytics in research and development. Examples for biospecific assays are hybridisation assays, immuno assays and receptor-ligand assays.
Hybridisation assays use the specific base-pairing for the molecular detection of nucleic acid analytes e.g. RNA and DNA. Hence, oligonucleotide probes with a length of 18 to 20 nucleotides may enable the specific recognition of a selected complementary sequence e.g. in the human genome. Another assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of desoxynucleotide triphosphates in several cycles.
As described above, before the biological substances may be analysed in one of the above-mentioned assays or used for other processes, it has to be isolated or purified from biological samples containing complex mixtures of different components as e.g. proteinaceous and non-proteinaceous components. Often, for the first steps, processes are used which allow the enrichment of the component of interest, e.g. the nucleic acid. Frequently, these are contained in a bacterial cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human blood cell or a plant cell. The component of interest can also be called a “target component”.
To release the contents of said cells or particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls of such organisms. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during lysis is that other enzymes degrading the target component, e.g. desoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the component of interest during lysis. These degrading enzymes may also be present outside of the cells spatially separated in different cellular compartments before lysis and come into contact with the target component after lysis. Other components released during this process may be e.g. endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.
There are a variety of means to tackle this problem mentioned-above. It is common to use chaotropic agents as e.g. guanidinium thiocyanate or anionic, cationic, zwitterionic or non-ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases which rapidly degrade these enzymes or unwanted proteins. However, this may produce another problem as the said substances or enzymes can interfere with reagents or components in subsequent steps.
Enzymes which can be advantageously used in such lysis or sample preparation processes mentioned-above are enzymes which cleave the amide linkages in protein substrates and which are classified as proteases, or (interchangeably) peptidases (See Walsh, C., Enzymatic Reaction Mechanisms (1979) chapter 3, W. H. Freeman and Company, San Francisco). Proteases which have been used are e.g. alkaline proteases (WO 98/04730) or acid proteases (U.S. Pat. No. 5,386,024). The protease which is widely used for sample preparation for the isolation of nucleic acids is proteinase K from Tritirachium album (see e.g. Sambrook, J., et al.: Molecular Cloning (1989) Cold Spring Harbor University Press, NY, USA) which is active around neutral pH and belongs to a family of proteases known to the person skilled in the art as subtilisins. A subtilisin is a serine protease produced by Gram-positive bacteria or fungi.
In the next steps of the sample preparation which follow on the lysis step, the target component is further enriched. If the target component is a nucleic acid, the target nucleic acid is normally extracted from the complex lysis mixtures before it is used in a probe-based assay.
There are several methods for the extraction of nucleic acids:                sequence-dependent or biospecific methods, e.g.:        affinity chromatography        hybridisation to immobilised probes        sequence-independent or physico-chemical methods, e.g.:        liquid-liquid extraction with e.g. phenol-chloroform        precipitation with e.g. pure ethanol        extraction with filter paper        extraction with micelle-forming agents as cetyl-trimethyl-ammonium-bromide        binding to immobilised, intercalating dyes, e.g. acridine derivatives        adsorption to silica gel or diatomic earths        adsorption to magnetic glass particles (MGP) or organo silane particles under chaotropic conditions.        
USPTO Backgrounds
USPTO Backgrounds