Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-29
2004-04-27
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06727067
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. §119 of foreign applications EP Application No. 00123728-8, filed Oct. 31, 2000, and EP Application No. 01106308-8, filed Mar. 15, 2001, the contents of which are hereby incorporated by reference in their entireties.
This invention relates to a method for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample using a protease from a Bacillus strain. The invention further relates to a method for the analysis of a (at least one) target nucleic acid component of a mixture of non-proteinaceous components, which comprise nucleic acids, and proteinaceous components whereby the mixture is derived from a biological sample comprising the steps of incubating the mixture with a (at least one) protease from a Bacillus strain, optionally amplifying the (at least one) target nucleic acid component, and determining or detecting the (at least one) target nucleic acid component.
BACKGROUND OF THE INVENTION
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 non-proteinaceous material such as nucleic acids. 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 the lysis is that other enzymes degrading the non-proteinaceous component of interest, 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 the cells or may have been spatially separated in different cellular compartiments before the lysis and come now into contact with the component of interest. 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, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3). Proteases which have been used in the prior art are e.g. alkaline proteases (W098/04730) or acid proteases (U.S. Pat. No. 5,386,024). The protease which is widely used in the prior art for sample preparation for the isolation of nucleic acids is proteinase K from
Tritirachium album
(see e.g. Sambrook et al., 1989) 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.
Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, referred to as subtilisin. A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, in which there is an essential serine residue at the active site (White, Handler, and Smith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, N.Y., pp. 271-272). The serine proteases have molecular weights in the 25,000 to 30,000 Da (Dalton) range. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. The alternative term, alkaline protease, reflects the high pH optimum of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev. 41: 711-753).
A wide variety of subtilisins have been identified (see e.g. Kurihara et al., 1972, J. Biol. Chem. 247: 5629-563 1; Stahl and Ferrari, 1984, J. Bacteriol. 158: 411-418; Vasantha et al., 1984, J. Bacteriol. 159: 811-819, Jacobs et al., 1985, Nucl. Acids Res. 13: 8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366: 421-430; Svendsen et al., 1986, FEBS Lett 196: 228-232; Meloun et al., 1985, FEBS. Lett. 183: 195-200) including proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366: 584-492). Subtilisins are well characterized by their primary as well as by their tertiary structure (see e.g. Kraut, 1977, Ann. Rev. Biochem. 46: 331-358; Kurihara et al., 1972, J. Biol. Chem. 247: 5629-5631; Stahl and Ferrari, 1984, J. Bacteriol. 158: 411-418; Vasantha et al., 1984, J. Bacteriol. 159: 811-819; Jacobs et al., 1985, Nucl. Acids Res. 13: 8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366: 421-430; Svendsen et al., 1986, FEBS Lett. 196: 228-232; Meloun et al., 1985, FEBS Lett. 183: 195-200; Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366: 485-492).
In connection with this invention the amino acid and DNA sequences of two further serine proteases are of particular interest. These proteases were derived from two Bacillus lentus variants, 147 and 309, which have been deposited with NCIB and designated the accession Nos. NCIB 10147 and NCIB 10309, respectively (see WO89/06279 and U.S. Pat. No. 3,723,250). For convenience the proteases produced by these strains are designated subtilisin 147 and subtilisin 309, respectively, and the genes encoding these proteins are referred to as the subtilisin 147 and 309 genes. The disclosure of these sequences can be found in WO89/0627
Meier Thomas
Russman Eberhard
Schmuck Ranier
Staepels Johnny
Wehnes Uwe
Horlick Kenneth R.
Pennie & Edmonds LLP
Roche Molecular Systems Inc.
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