Title of Invention	METHOD FOR DETECTING PROKARYOTIC DNA FROM BODY FLUIDS IN VITRO
Abstract	A method is described for enriching procaryotic DNA, said method including the steps of contacting at least one procaryotic DNA with at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs, and separating the protein/polypeptide-DNA complex. Moreover, the application relates to a kit for carrying out said method.

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"METHOD FOR DETECTING PROKARYOTIC DNA FROM BODY FLUIDS IN VITRO" The invention relates to a method of enriching prokaryotic DNA as well as to a kit for carrying out said method. Infections caused by bacteria are one of the most frequent causes of inflammatory diseases. For the prognosis of the clinical course as well as, in particular, for timely selection of suitable therapeutic measures, early detection of the bacterial pathogens is of decisive importance. In the detection of bacterial pathogens, use is made, above all, of different methods of cultivating cells. However, methods of molecular biology which are based on the detection of pathogen-specific nucleic acids have also become more important recently. In addition to the high specificity of these methods, mention must be made of the little time required as an essential advantage over conventional methods. However, the sensitivity of the detection of prokaryotic DNA directly from body fluids and from .test material that has not been pre treated has hitherto been much too low as compared to the culture of microorganisms. An amount of nucleic acids of bacteria sufficient _to _detect pathogens directly from the test material that has not been pre-treated is achieved, if at all, in the region of the 16S-mRNA molecules. However, this requires that the bacteria to be detected be present in the metabolic phases and express sufficient 16S-mRNA. This is usually not the case, in particular in patients who are subject to antibiotic therapy. Moreover, certain prokaryotic factors of bacteria are not expressed every time, although the corresponding genes are present in the bacterial genome. Therefore, the detection of the prokaryotic factors and resistance of bacteria at the chromosomal level is indispensable for the diagnosis of septic disease states. -2- This applies even more because, at this level, a distinction can also be made between pathogenic and commensal bacteria. Most frequently, the detection of pathogen-specific nucleic acids is effected by amplification of the procaryotic DNA by means of the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), respectively. The high specificity and fast availability of the results is contrasted by the susceptibility to interference or by strongly inhibiting factors of clinical samples. In a conventional PCR detection method, successful detection of pathogens in the blood requires isolation of total DNA from at least 1 to 5 ml of blood. However, the total DNA concentration is then too high to be employed directly in a PCR reaction. Things are different with regard to the blood culture for detection of sepsis pathogens. In this case, the lower detection limit is less than 10 bacteria per ml. This detection limit is presently achieved only by PCR protocols whose target sequence is in the 16S-RNA region and which are therefore dependent on the expression of said target sequence. Greater diagnostic reliability can be expected of PCR protocols which have their target sequences in the chromosome of the microorganisms. The expression behavior of different genes can be considerably changed or limited, especially under the influence of an ongoing antibiotic therapy, even if the antibiotic used is ultimately not effective. This situation is often found particularly in intensive therapy wards, where most patients receive antibiotic treatment, thus not allowing to grow any relevant bacteria from blood cultures or other samples for this reason. Due to insufficient sensitivity, the detection of pathogen-specific nucleic acids, wjthout^an amplification step by direct detection of procaryotic DNA (probe technique, FISH technique), is of diagnostic importance only at a sufficiently high germ number in the test material. The essential problems of the detection of procaryotic DNA for identification of bacterial pathogens in body fluids consist, beside PCR-inhibiting ingredients in the test material, mainly in the excess of eucaryotic DNA versus procaryotic DNA. In this connection, competitive processes in DNA analysis as well as the tow quantity of procaryotic DNA can be regarded as a hindrance to a qualitative and quantitative detection of pathogens. The usual methods of DNA isolation enrich the total DNA of a body fluid so that the ratio of host DNA to microbial DNA may be between 1:10-6and 1:10-8. This difference makes the. _ _ difficulty in detecting microbial DNA in body fluids quite clear. -3- Tfcerefore, it is an object of the present invention to provide a method of isolating and/or if enriching microbial DNA, in test samples having a high content of eucaryotic DNA from patients with infections, for quick and easy detection of pathogens, said detection enabling early diagnosis of infections caused by bacterial pathogens. According to the invention, this object is achieved by a method of eririching procaryotic DNA, _ comprising the steps of ^\_^/ a) contacting at least one procaryotic DNA in solution with at least one protein or polypeptide which is capable of specifically binding to procaryotic DNA, thus forming a protein or pofy^p^io^jpj^compjexjjnd b) separating said complex. In this case, the term procaryotic DNA relates to both viral and bacterial DNA. Said DNA may be purified and dissolved again or may be present directly in the original source (e.g. body fluid, such as blood, serum, etc.). Separation may be effected by means of different methods of isolating or enriching DNA protein complexes or DNA polypeptide complexes that are well-known to the person skilled in the art. In doing so, use will be made preferably of methods in which the DNA-binding protein is immobilized to a carrier matrix in order to enrich the DNA from the sample solution. According to a preferred embodiment, the separation is followed by a step of separating the DNA and the protein/polypeptide. This may be effected, for example, by conventional methods of DNA purification which are known to the person skilled in the art. In the most simple case, the separation is based on the change in pH value or in the salt concentration (e.g. to 1 M NaCI) of the medium/buffer or on the addition of chaotropic reagents, etc.; i.e. suitable parameters which lead to the separation of thTp^eirvOTlA^compiex. Such methods are known to the person skilled in the art. According to a further preferred embodiment, the protein or the polypeptide is coupled to a carrier. This embodiment represents a particularly simple way of enriching procaryotic DNA, because the separation from the solution is particularly easy, for example by means of physical removal (e.g. by centrifugation) of the charged carriers) from the solution. As the solution of the procaryotic DNA, any suitable solvent is basically suitable. However, i the method is particularly useful for enriching procaryotic DNA from solutions which contain „ -4- different biomolecular species, in particular different types of DNA. The invention preferably relates to a method of separating and enriching procaryotic or viral DNA and eucaryotic DNA from a mixture of procaryotic or viral DNA. In doing so, for example, the procaryotic DNA which is present in body fluids is separated from the eucaryotic DNA, by specific binding to the protein or to the polypeptide, and enriched. The procaryotic DNA enriched in this way facilitates detection of procaryotic pathogens with the help of molecular biology methods and can contribute to the diagnosis of diseases caused by pathogenic pathogens. In particular, the embodiment according to which the DNA-binding protein or polypeptide is immobilized to the surface of a carrier is suitable for adsorption of procaryotic DNA from body fluids, preferably from blood. Moreover, this approach allows removal of microbtal DNA, which is present in blood or other body fluids, from said fluids. The body fluid (e.g. whole blood, serum or liquor) purified in this way from the mtcrobial DNA, which is also capable in itself of initiating severe inflammatory reactions in patients, can then be fed back into the body. Body fluids in the sense of the invention are understood to be all fluids originating from the body of a mammal, including humans, in which disease pathogens may occur, such as blood, urine, liquor, pleural, pericardial, peritoneal as well as synovial fluids. The description of the invention referring to human blood is not to be construed as limitative, but only as an exemplary application. Proteins or polypeptides in the sense of the invention are understood to be all eucaryotic and procaryotic proteins which are capable of specifically binding procaryotic DNA. Proteins or polypeptides which are capable of specifically binding non-methylated CpG-motifs are particularly suitable for this purpose. Bacterial pathogens are preferably understood to be pathogens of sepsis, but also any other bacterial pathogens of infections. They may differ from commensal pathogens, which are sometimes also found in test samples from patients, but do not have any pathogenic significance. In isolating the total DNA from infected body liquids, the ratio of host-DNA to pathogen-DNA may be, in many cases, 1:10-6 to 1:1-8 and less. Through the specific binding of procaryotic DNA to the protein or polypeptide having such selective properties, the method according to the invention enables enrichment by 3 exponential units and more. -5- The protein or the polypeptide may be coupled directly or indirectly to the carrier. The type of coupling depends on the carrier and the carrier material. Suitable carriers include, in particular, membranes, microparticles and resins, or similar materials for affinity matrices. Suitable materials for binding of the protein or of the polypeptide, as well as - depending on the type of material - for carrying out such binding, are well-known to the person skilled in the art. For indirect coupling, such specific antibodies against the protein or polypeptide are suitable, for example, which are in turn bound to the carrier by known methods. One application of the method according to the invention consists in enriching procaryotic DNA. A further application consists in the separation of procaryotic DNA from a mixture of eucaryotic and jxgcaryotic DNAJ>yj3[nding of the procaryotic DNA to a specific protein or polypeptide which has been immobilized to a matrix. The mixture of the body's own DNA and procaryotic DNA is contacted with the affinity matrix by means of suitable methods and, in doing so, the procaryotic DNA is bound to the immobilized protein; the eucaryotic DNA passes, for example, through a separating column and may be collected separately. Affinity matrices may be, for example, ^polymeric polysaccharides, such as agaroses, other fSiopolymers, synthetic polymers, or carriers having a silicate~b"acRBonersuch as porous glasses or other solid or flexible carriers on which the DNA-binding protein or polypeptide is immobilized. After separation of procaryotic DNA from eucaryotic DNA has been effected, the affinity matrix is rinsed with a suitable reagent, so that either the binding protein with the coupled procaryotic DNA is separated from the matrix and/or the procaryotic DNA is separated from the binding protein and is available for further process steps in a sufficient amount. A further application of the method according to the invention consists in the separation and enrichment of procaryotic DNA from eucaryotic DNA by binding of the procaryotic DNA to a specific protein which has been immobilized on microparticles. In this connection, all microparticles which allow the DNA-binding protein or polypeptide to be immobilized are suitable. Such microparticles may consist of latex, plastics (e.g. styrofoam, polymer), metal or ferromagnetic substances. Furthermore, use may also be made of fluorescent microparticles, such as those available from the Luminex company, for example. After the procaryotic DNA has been bound to the proteins immobilized on microparticles, said microparticles are separated from the mixture of substances by suitable methods, such as filtration, centrifugation, precipitation, sorting by measuring the intensity of fluorescence, or by magnetic methods. After separation from the microparticles, the procaryotic DNA is available for further processing. -6- Another application of the method according to the invention consists in the separation and enrichment of procaryotic DNA from eucaryotic DNA by binding of the procaryotic DNA to a specific protein or polypeptide, which is subsequently separated from other ingredients of the mixture by electrophoresis. A further application of the method according to the invention consists in the separation and enrichment of procaryotic DNA from eucaryotic DNA by binding of the procaryotic DNA to the protein or polypeptide. Said protein is subsequently bound to corresponding antibodies. The antibodies may be bound to solid or flexible substrates, such as glass, plastics, silicon, microparticles, membranes, or may be present in solution. After binding of the procaryotic DNA to the protein or the polypeptide and binding of the latter to the specific antibody, separation from the substance mixture is effected by methods known to the person skilled in the art. As protein or polypeptide, any protein or polypeptide is particularly suitable which binds procaryotic DNA with non-methylated CpG motifs, for example. For this purpose, specific antibodies or antisera against procaryotic DNA are suitable, for example. Their preparation and isolation are known to the person skilled in the art. Procaryotic DNA differs from eucaryotic DNA, for example, by the presence of non-methylated CpG motifs. Thus, the protein/polypeptide is conveniently a protein which specifically recognizes and binds non-methylated CpG motifs. Conveniently, this also includes a specific antibody or a corresponding antiserum. According to a further preferred embodiment, the protein or polypeptide is a protein or polypeptide encoded by the TLR9 gene or by the CGBP gene. This embodiment of the invention is based on the finding that eucaryotic DNA and procaryotic DNA differ in their content of CpG motifs. In the procaryotic DNA, cytosine-guanosine-dinucleotides (CpG motifs) are present in an excess of 20 times that of eucaryotic DNA. In procaryotic DNA, these motifs are non-methylated, whereas they are methylated for the most part in eucaryotic DNA, which further enhances the difference. Non-methylated CpG motifs are non-methylated deoxycytidylate-deoxyguanylate-dinucleotides within the procaryotic genome or within fragments thereof. Secondly, this preferred embodiment of the invention is based on the finding that there are proteins or polypeptides which bind specifically to non-methylated CpG motifs of the DNA. The binding property of these proteins/polypeptides is used, according to the invention, in -7- order to bind procaryotic DNA, on the one hand, and thus to enrich it, on the other hand, from a sample mostly containing eucaryotic DNA. An application for isolating cDNA, which uses the presence of methylated CpG motifs in eucaryotic DNA was described by Cross et al. Nature Genetics 6 (1994) 236-244. The immunostimulatory application of single-stranded oligodeoxyribonucleotides (ODN) with the corresponding CpG motifs has been shown several times (Hacker et al., Immunology 105 (2002) 245-251, US 6,239,116). As recognition molecules of the procaryotic CpG motifs, two receptor proteins have been identified so far. Toll-Iike-receptor 9 is known from WO 02/06482 as a molecule recognizing non-methylated CpG motifs. Voo et al. Molecular and Cellular Biology (2000) 2108-2121 describe a further receptor protein, i.e. the human CpG-binding protein (hCGBP), which is used in an analytic approach as a recognition molecule for detecting non-methylated CpG motifs in procaryotic DNA. In both publications, the CpG-binding proteins are not used for isolating or enriching procaryotic DNA. A protein or polypeptide which is encoded by cDNA having a sequence with a homology of at least 80%, preferably at least 90%, and particularly preferably at least 95%, to the sequence according to gene bank access no.: NM-014593 (version NM-014593 1, Gl: 7656974; NCBI database) is particularly suitable. These are proteins or polypeptids which correspond to CGBP or are derived therefrom and which specifically recognize and bind CpG motifs. According to a further preferred embodiment, the protein or polypeptide is encoded by cDNA having a sequence with a homology of at least 80%, preferably at least 90%, to the sequence according to gene bank access no. AB045180 (coding sequence of the TLR9 gene; NCBI database, version AB045180.1; Gl: 11761320) or a fragment thereof, preferably cDNA having a homology of at least 80%, particularly preferably 90%, to transcript variant A (gene bank access no. NM-138688; version NM-017442.1; Gl: 20302169; NCBI database) or transcript variant B (gene bank access no. NM-017442; version NM-138688.1; Gl: 20302170; NCBI database). Moreover, the invention relates to a method of purifying body fluids to remove procaryotic DNA. In this connection, it is convenient for the separation to be effected extracorporally, under sterile conditions, to allow the body fluids to be fed back into the body again, so that the body's own immune system is assisted in eliminating infections by removing the procaryotic DNA contained in said body fluids. -8- Any suitable chemical, mechanical or electrochemical processes may be considered for the extracorporal removal of procaryotic DNA from body fluids. Further, the combination with other extracorporal therapeutic methods, such as hemoperfusion, heart-lung machine or endotoxin absorbers, represents a further convenient application. This enumeration does not represent a limitation of the methods. According to a particularly preferred embodimet -the invention relates to a method of detecting procaryotic DNA. In this case, the eniichment of the" procaryotic DNA-is-followed' by a step of amplifying_said procaryotic DNA, for which all common methods of amplification are suitable (PCR, LCR; LM-PCR, etc.). Moreover, the invention relates to a kit for enriching procaryotic DNA by means of one of the above-described methods, said kit containing at least the protein/polypeptide, preferably further reagents suitable to carry out said method. According to a preferred embodiment, said kit contains, in addition to the protein/polypeptide, at least one set of primers, which are suitable to amplify genomic DNA of certain procaryonts under standard conditions. The invention has the advantage that, by specific binding of non-methylated procaryotic DNA rich in CpG motifs to proteins with specific affinity for such structures, procaryotic DNA from the total DNA of an infected host is successfully concentrated and thus the sensitivity of detection of pathogen DNA in body fluids is strongly enhanced. The possibilities of separating procaryotic DNA from eucaryotic DNA using a specifically binding protein are no more time-consuming than known methods of isolating total DNA. However, the following detection can then be effected only via a PCR reaction. A nested PCR will not be required in most cases, which makes it possible to save a considerable amount of time in diagnostics. The invention will be explained in more detail below by means of examples, without limiting it thereto. Fig. 1 shows the PCR of streptococci-DNA in human blood, and Fig. 2 shows the nested PCR with the PCR products according to Fig. 1. -9-Example 1: Prior art method of detection Fresh, heparinized human blood, which contains streptococcus pyogenes with 103/ml colony-forming units as pathogens, is used for detection of pathogens. The DNA is isolated by means of absorption to DNA-binding matrix using commercial kits for isolation of total DNA from body fluids according to modified instructions from the manufacturer. For this purpose, 200 µl of the total lysis buffer, which contains proteinase K and SDS, is added to 100 µl of infected blood in Eppendorf tubes. The mixture is incubated at 37°C for 30 min. and then heated to 95°C for 20 min. After cooling, 20ug of mutanolysine are added and incubated at 37°C for another 60 min. After centrifugation, the supernatant is applied to the centrifugal columns using DNA-binding matrix and the DNA is purified according to the manufacturer's instructions. The purified DNA is placed in a final volume of 100pl of 0.01 mol tris buffer, pH 7.5, or in an equal amount of elution buffer from the manufacturer. For detection of pathogens, primers are selected to identify the streptolysin o gene (slo). 1. PCR. Amplification of a 465 bp fragment Forward primer 1: 5'-AGCATACAAGCAAATTTTTTACACCG Reverse primer 2: 5'-GTTCTGTTATTGACACCCGCAATT Primer concentration 1mg/ml Starting material: 5 µl isolated DNA 0.5 µl primer fw 1 0.5 µl primer rv 2 14 µl aquadest total 25 µl in Ready to go Kit (Amersham-Biosciences) Reaction: 1 x 5 min 95 °C 40 cycles each at 30 sec. 95°C 30 sec. 51 °C 3 min 72°C 1 x 7 min 72°C The results of the PCR of streptococci-DNA in human blood are shown in Fig. 1. 10 µl of the 25 µl of starting material were separated. 1) PCR starting material containing 5 µl template DNA; 2) starting material containing 5 µl template, at a dilution of 1:10. 3) positive control: 0.2 µl of streptococci-DNA as template in the absence of eucaryotic DNA from blood. ST) molecular weight standard -10- Result: The primary PCR does not result in a visible PCR product. Therefore, a 2. PCR (nested PCR) was carried out as below. 2. PCR (nested): Amplification of a 348 bp fragment contained in the above slo-fragment. Forward primer 3: 5'- CCTTCCTAATAATCCTGCGGATGT-3' Reverse primer 4: 51- CTGAAGGTAGCATTAG TCTTTGATAACG-31 Primer concentration: 1mg/ml Starting material: 5 ul from PCR1, sample 1, Fig. 1 0.5 ul primer fw 1 0.5 \J\ primer rv 2 14 ul aquadest total 25 ul in Ready to go Kit (Amersham-Biosciences) Reaction: 1 x 5 min 95°C 50 cycles each at 30 sec. 95°C 30 sec. 54°C 3 min 72°C 1 x 7 min 72°C Fig. 2 shows the nested PCR with the PCR products according to Fig. 1 as template. The samples correspond to those of Fig. 1. Result: In the nested PCR, the desired slo-DNA fragment is amplified at a pathogen number of 100 streptococci cells per 100 ul blood (sample 1). At 5 ul template DNA in the 1st PCR (Fig. 1), this corresponds to about 5 to 10 template molecules. At a dilution of 1:10 (sample 2), sensitivity is exhausted (0.5 to 1 template molecules). Example 2: Carrying out the method according to the invention The DNA is dissolved from a cell lysate as described above for the previous PCR methods. The difference is that between 1 ml and 5 ml of test material are employed. Three milliliters of fresh, heparinized or citrate-added human blood, which contains streptococcus pyogenes with 102/ml colony-forming units as pathogens, is used for detection of pathogens. The DNA is isolated by means of lysis buffers which contain SDS and proteinase K, using commercial kits to isolate total DNA from body fluids according to modified instructions from the manufacturer. For this purpose, 6 ml of the total lysis buffer. -11 - which contains proteinase K and SDS, is added to 6 ml of infected blood. The mixture is incubated at 37°C for 30 min. and then heated to 95°C for 20 min. After cooling, 200 µg of mutanolysine are added and incubated at 37 °C for another 60 min. After centrifugation, the mixture is precipitated with ethanol at a final concentration of 70 %, and upon centrifugation, the pellet is washed with 2 ml of 70 % ethanol. The ethanol residue is removed in a vacuum centrifuge and the precipitated DNA is collected in 500 µl TE buffer. The DNA is then applied to a column which contains 0.5 ml of sepharose and is immobilized on the 1 mg of TLR9. The column is washed with 5 volumes of TE buffer. Elution is carried out with chaotropic ions at a high concentration, e.g. with 0.7 ml of a 6 mole NaJ or KSCN solution. This eluate can then be applied directly to a commercial DNA-isolating centrifugal column, and the CpG-enrtched DNA may be isolated according to instructions, as in the initial example, to a small volume of between 20 µl and 100 µl and employed for further analysis, such as pathogen PCR. 12 WE CLAIM: 1. Method for detecting prokaryotic DNA from body fluids in vitro, comprising the steps of: a) contacting at least one prokaryotic DNA in solution with at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs, b) separating the protein/polypeptide-DNA complex from the solution, c) detecting the prokaryotic DNA by means of methods of molecular biology 2. Method as claimed in claim 1, wherein the separation is followed by a step of separating the DNA and the protein/polypeptide 3. Method as claimed in one of the preceding claims, wherein the prokaryotic DNA is detected by means of amplification 4. Method as claimed in one of the preceding claims, wherein the prokairyotlc DNA is detected by means of probe technology 5. Method for purifying body fluids from prokaryotic DNA in vitro comprising the steps of: a) contacting at least one prokaryotic DNA in a body fluid with at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs, b) separating the protein/polypeptide-DNA complex from the body fluid 6. Method as claimed in one of the preceding claims, wherein the protein/ polypeptide is coupled to a carrier. 7. Method as claimed in claim 6, wherein the protein/ polypeptide is directly coupled to a carrier. 8. Method as claimed in claim 6, wherein the protein/ polypeptide is coupled to a carrier via an antibody. 9. Method as claimed in one of the claims 6 to 8, wherein the carrier is provided as a matrix,microparticles or a membrane. 10. Method as claimed in one of claims lor 2, wherein the separation takes place by means of an antibody or antiserum directed against the protein/polypeptide 11. Method as claimed in claim 1, wherein the separation is carried out by means of electrophoresis 12. 12. Method as claimed in one of the preceding claims, wherein the protein/ polypeptide is an antibody directed against non-methylated CpG motifs,or is a corresponding antiserum. 13. Method as claimed in one of the claims 1 to 11, wherein the protein/polyp eptide is encoded by the TLR9 gene or by the CGBP gene.. 14. Method as claimed in claim 13, wherein the protein/polypeptide is encoded by cDNA with a sequence which is at least 80% and preferably at least 90% homologous to the following sequence ggcacgagag acggcggcgc ctgaggggtc ttgggggctc baggccggcc acctactggt ttgcagcgga gacgacgcat ggggcctgcg caataggagt acgctgcctg ggaggcgtga ctagaagcgg aagtagttgt gggcgccttt gcaaccgcct gggacgccgc cgagtggtct gtgcaggtbc gcgggtcgct ggcgggggtc gtgagggagt gcgccgggag cggagatatg gagggagatg gttcagaccc agagcctcca gatgccgggg aggacagcaa gtccgagaat ggggagaatg cgcccatcta ctgcatctgc cgcaaaccgg acatcaactg cttcatgatc gggtgtgaca actgcaatga gtggttccat ggggactgca tccggatcac cgagaagatg gccaaggcca tccgggagCg gtactgtcgg gagtgcagag agaaagaccc caagctagag attcgctatc ggcacaagaa gtcacgggag cgggatggca atgagcggga cagcagtgag ccccgggatg aggg-tggagg gcgcaagagg cctgtccctg atccaaacct gcagcgccgg gcagggtcag ggacaggggt tggggccatg cttgctcggg gctctgcttc gccccacaaa tcctctccgc agcccttggt ggccacaccc agccagcatc accagcagca gcagcagcag atcaaacggt cagcccgcat gtgtggtgag tgtgaggcat gtcggcgcac tgaggactgt ggtcactgtg attitctgtcg ggacatgaag aagttcgggg gccccaacaa gatccggcag aagtgccggc tgcgccagtg ccagctgcgg gcccgggaat cgtacaagta cCtcccttcc tcgctctcac cagtgacgcc ctcagagtcc ctgccaaggc cccgccggcc actgcccacc caacagcagc cacagccatc acagaagtta gggcgcatcc gtgaagatga gggggcagtg gcgtcstcaa cagtcaagga gcctcctgag gctacagcca cacctgagcc actctcagat gaggacctac ctctggatcc tgacctgtat caggacttct gtgcaggggc ctttgatgac aatggcctgc cctggatgag cgacacagaji gagtccccat tccCggaccc cgcgctgcgg aagagggcag tgaaagtgaa gcatgtgaag cgfccgggaga agaagtctga gaagaagaag gaggagcgat acaagcggca tcggcagaag cagaagcaca aggataaatg gaaacaccca gagagggctg atgccaagga ccctgcgtca ctgccccagt gcctggggcc cggctgtgtg cgccccgccc agcccagctc caagtattgc tcagaCgact gtggcatgaa gctggcagcc aaccgcatct acgagatcct cccccagcgc ntccagcagt ggcagcagag cccttgcatt gctgaagagc acggcaagaa gctgctcgaa cgcattcgcc gagagcagca gagtgcccgc acccgccttc aggaaatgga acgccgattc t;atgagcttg aggccaCcat Cctacgtgcc aagcagcagg ctgtgcgcga ggatgaggag agcaacgagg gtgacagtga tgacacagac ctgcagatct tctgtgtttc ctgtgggcac cccatcaacc cacgtgttgc cttgcgccac cgggagcgct gctacgccaa gtatgagagc cagacgtcct ttgggtccat gtaccccaca cgcattgaag gggccacacg actcttctgt: gatgtgtata atcctcagag caaaacatac tgtaagcggc tccaggtgct gtgccccgag cactcacggg accccaaagt gcqagctgac gaggtatgcg ggtgccccct tgtacgtgal; gtctttgagc tcacgggtga cttctgccgc ctgcccaagc gccagtgcaa tcgccattac cgctgggaga agctgcggcg tgcggaagtg gacttggagc gcgtgcgtgb gtggtacaag ctggacgagc tgtttgagca ggagcgcaat gtgcgcacag ccatgacaaa ccgcgcggga fctgctggccc tgatgctgca ccagacgatc cagcacgatc ccctcactac cgacctgcgc: ticcagtgccg accgctgagc cccctggccc ggacccctta aaccctgcat tccagatggg ggagccgccc ggtgcccgtg tgtccgttcc tccactcatc tgtttctccg gttctccctg .gcccatcca ccggttgacc gcccatctgc ctttatcaga gggactgtcc ccgtcgacat: gttcagtgcc tggtggggct gcggagtcca ctcatccttg cctcctctcc ctgggttttg titaataaaat tttgaagaaa cc and is capable of specifically binding to non-methylated CpG motifs. 15. Method as claimed in claim 13, wherein the protein/polypeptide is encoded by cDNA with a sequence which is at least 80% and preferably at least 90% homologous to the following sequence ccgctgcCgc ccctgtggga agggacctcg agtgtgaagc atccttccct gtagctgctg tccagtcCgc ccgccagacc ctctggagaa gcccctgccc cccagcatgg gtttctgccg cagcgccctg cacccgctgt ctctcctggt gcaggccatc atgctggcca tgaccctggc cctgggtacc ttgcctgcct Ccctaccctg tgagctccag ccccacggcc tggtgaactg caactggctg ttcctgaagt ctgtgcccca cttctccatg gcagcacccc gtggcaacgt caccagcctt tccttgC-ccC ccaaccgcat ccaccacctc cacgattctg actttgccca cctgcccagc ctgcggcatc tcaacctcaa1 gtggaactgc ccgccggttg gcctcagccc catgcacttc ccctgccaca tgaccatcga gcccagcacc ttcttggctg tgcccaccct ggaagagcta aacctgagct acaacfiacat catgactgtg cctgcgctgc ccaaatccct catatccctg tccctcagcc atacccLacat cctgatgcta gactctgcca gcctcgccgg ccCgcatgcc ctgcgcCtcc tattcatgga cggcaactgt tattacaaga acccctgcag gcaggcactg gaggtggccc cgggtgccct ccttggcctg ggcaacctca cccacctgtc actcaagtac aacaacctca ctgtggtgcc ccgcaacctg ccCtccagcc tggagtatct gctgttgtcc tacaaccgca tcgtcaaact ggcgcctgag gacctggcca atctgaccgc cctgcgtgtg ctcgatgtgg gcggaaattg ccgccgctgc gaccacgctc ccaacccctg catggagtgc cctcgtcact tcccccagct acatcccgat accttcagcc acctgagccg tcttgaaggc ctggtgttga aggacagttc tctctcctgg ctgaatgcca gttggttccg tgggctggga aacctccgag tgctggacct gagtgagaac ttcctctaca aatgcatcac taaaaccaag gccttccagg gcctaacaca gctgcgcaag cttaacctgt ccttcaatta ccaaaagagg gtgtcccttg cccacctigtc tctggcccct tccttcggga gcctggtcgc1 cctgaaggag ctggacatgc acggcatictt cttccgctca ctcgatgaga ccacgctccg gccactggcc cgcctgccca tgctccagac tctgcgtctg cagatgaact tcatcaacca ggcccagctc ggcatcttca gggccttccc tggcctgcgc tacgtggacc tgtcggacaa ccgcatcagc ggagcttcgg agctgacagc caccatgggg gaggcag-atg gaggggagaa ggtctggctg cagcctgggg accttgctcc ggccccagtg gacactccca gctctgaaga cttcaggccc aactgcagca cecteaactt caccttggat ctgtcacgga acaacctggt gaccgtgcag ccggagatgt tcgcccagct ctcgcacctg cagtgcctgc gcctgagcca caactgcatc tcgcaggcag tcaatggctc ccagttcctg ccgctgaccg gtctgcaggt gctagacctg tcccacaata agctggacct ctaccacgag cactcattca cggagctacc acgactggag gccctggacc tcagctacaa cagccagccc tttggcatgc agggcgtggg ccacaacttc agcttcgtgg ctcacctgcg caccctgcgc cacctcagcc tggcccacaa cascatccac agccaagtgt cccagcagct ctgcagtacg tcgctgcggg ccccggactt cagcggcaat gcactgggcc atatgtgggc cgagggagac ctctatctgc acttcttcca aggcctgagc ggtttgatct ggctggactC gtcccagaac cgcctgcaca ccct&ctgcc ccaaaccctg cgcaacctcc ccaagagcct acaggtgctg cgtctccgtg'acaattacct ggccttcttt aagtggtgga gcctccactt cctgcccaaa ctggaagtcc tcgacctggc aggaaaccag ctgaaggccc tgaccaatgg cagcctgcct gctggcaccc ggctccggag gctggatgtc agctgcaaca gcatcagctt cgtggccccc ggcttctttt ccaaggccaa ggagctgcga gagcCcaaCc ttagcgccaa cgccctcaag acagtggacc actcctggcc tgggcccctg gcgagtgccc tgcaaabact agatgtaagc gcdaaccctc tgcactgcgc ctgtggggcg gcctttaCgg acCtcctgct ggagg-tgcag gcCgccgtgc ccggtctgcc cagccgggtg aagtgtggca gtccgggcca gctccagggc ctcagcatct ttgcacagga cctgcgcctc tgcctggaCg aggccctctc ctgggactgt ttcgccctct cgctgctggc tgtggccctg ggcctgggtg tgcccatgct gcatcacctc tgtggctggg acctctggta ctgctcccac ctgtgcctgg ccCggcttcc ctggcggggg cggcaaagtg ggcgagabga ggatgccctg cccCacgatg ccttcgtggt cttcgacaaa acgcagagcg cagtggcaga ctgggcgtac aacgagcttc gggggcagct ggaggagtgc cgbgggcgct gggcactccg cctgtgcctg gaggaacgcg actggctgcc tggcaaaacc ctctttgaga acctgtgggc ctcggcctat ggcagccgca agacgctgtt tgtgctggcc cacacggacc gggtcagtgg tctcttgcgc gccagcttcc tgctgg-ccca gcagcgcctg ctggaggacc gcaaggacgt cgtggtgctg gtgatcctga gccctgacgg ccgccgcccc cgctacgtgc ggctgcgcca gcgcctctgc cgccagagtg tcctcctctg gccccaccag cccagtggtc agcgcagctt ctgggcccag ctgggcatgg ccctgaccag ggacaaccac cacCtctata accggaactt ccgccaggga cccacggccg aatagccgtg agccggaatc ctgcacggtg ccacctccac actcacctca cctctgc or a fragment thereof, preferably cDNA which is at least 80% and and preferably at least 90% homologous to transcript variant A of the following sequence ggaggtcttg tttccggaag atgttgcaag gctgtggtga aggcaggtgc agcctagcct cctgctcaag ctacaccctg gccctccacg catgaggccc tgcagaactc tggagatggt gcctacaagg gcagaaaagg acaagtcggc agccgctgtc ctgagggcac cagctgtggt gcaggagcca agacctgagg gtggaagtgt cctcttagaa tggggagtgc ccagcaaggt gtacccgcCa ctggtgctat ccagaattcc catctctccc tgctccctgc ctgagctctg ggccttagct cctccctggg cttggtagag gacaggtgtg aggccctcat gggatgtagg ctgtctgaga ggggagtgga aagaggaagg ggtgaaggag ctgtctgcca ctttjactatg caaatggcct ttgactcacg ggaccctgt:c ctcctcactg gggcagggt ggagtggagg gggagctact aggctggtat aaaaatctlia cttcctctat tctctgagcc gctgctgccc 16 ctgtgggaag ggacctcgag tgtgaagcab ccbtccctgt agctgctgtc cagtctgccc gccagaccct ctggagaagc ccctgccccc cagcatgggt ttctgccgca gcgccctgca cccgctgtct ctcctggtgc aggccatcat gctggccatg accctggccc tgggtacctt gcctgccttc ctaccctgtg agctccagcc ccacggccbg gtgaactgca actggctgtb ccbgaagtct gtgccccact tctccatgtjc agcaccccgt ggcaatgbca ccagcctttc cttgtcctcc aaccgcabcc accacctcca tgattctgac tttgcccacc bgcccagcct gcggcatctc aacctcaagt ggaactgcoc gccggttggc ctcagcccca tgcacttccc ctgccacatg accatcgagc ccagcacctt cttggctgtg cccaccctgg aagagcbaaa cctgagctac aacaacatca tgactgtgcc tgcgctgccc aaatccctca tatccctgtc cctcagccat accaacatcc tgatgctaga ctctgccagc ctcgccggcc tgcatgccct gcgcttccta ttcatggacg gcaactgtta ttacaagaac ccctgcaggc aggcactgga ggtggccccg ggtgccctcc ttggc:ctggg caacctcacc cacctgtcac tcaagtacaa caacctcact gtggtgcccc gcaacctgcc ttccagcctg gagtatctgc tgttgtccta caaccgcatc gbcaaactgg cgcctigagga cctggccaat ctgaccgccc tgcgtgtgct cgatgtgggc ggaaattgcc gccgctgcga ccacgctccc aacccctgca tggagtgncc tcgtcacttc ccccagctac atcccgatac cttcagccac ctgagccgtc ttgaaggcct ggtgbtgaag gacagttctc bctcctggct gaatgccagt tggttccgtg ggctgggaaa cctccgagtg ctggacctga gtgagaactt ccbcbacaaa tgcatcacba aaaccaaggc ctbccagggc ctaacacagc tgcgcaagct taacctgtcc Ctcaattacc aaaagagggt gtcctttgcc cacctgtctc tggccccttc ctbcgggagc cbggtcgccc tgaaggagct ggacatgcac ggcatcttct tccgctcact cgabgagacc acgctccggc cactggcccg cctgcccaLg ctccagactc ttjcgtctgca gatgaactbc atcaaccagg cccagctcgg catcttcagg gccttccctg gccbgcgcba cgtggaccbg tcggacaacc gcatcagcgg agcttcggag cbgacagcca ccatggggga ggcagatgga ggggagaagg tctggctgca gcctggggac cCtgctccgg ccccagtgga cactcccagc tctgaagact tcaggcccaa ctgcagcacc cbcaacttca ccttggabcb gtcacggaac aacctggtga ccgtgcagcc ggagatgbtt gcccagctct c*jcacctgca gbgcctgcgc ctgagccaca actgcatctc gcaggcagbc aatggcbccc agttcctgcc gctgaccggt ctgcaggtgc tagaccbgtc ccacaataag ctggacctct accacgagca ctcabtcacg gagctaccac gactggaggc cctggacctc agctacaaca gccagccctt tggcatgcag ggcgbgggcc acaacttcag cttcgtggct cacctgcgca ccctgcgcca ccbcagcctg gcccacaaca acabccacag ccaagtgbcc cagcagctcb ccagtacgtc gcbgcgggcc ctggacttca gcggcaatgc actgggccat atgtgggccg agggagacct cbatctgcac ttcttccaag gcctgagcgg tttgatctgg cbggacbtgb cccagaaccg cctgcacacc ctccbgcccc aaaccctgcg caacctcccc aagagccbac aggbgctgcg bcbccgtgac aattacctgg ccttctttaa gtggtggagc ctccacttcc tgcccaaact ggaagbcctc gacctggcag gaaaccagct gaaggccctg accaatggca gcctgcctgc bggcacccgg ctccggaggc bggatgtcag ctgcaacagc abcagctbcg tggcccccgg ctbcbtbtcc aaggccaagg agctgcgaga gctcaacctt agcgccaacg cccbcaagac: agtggaccac tcctggtttg ggcccctggc gagtgcccbg caaacactag aCgtaagcgc: caaccctctg cactgcgcct gtggggcggc ccttatggac ttccCgcbgg aggtgcaggt: tgccgtgccc ggtctgccca gccgggtgaa 17 gtgtggcagt ccgggccagc fcccagggcct: cagcatcttt gcacaggacc tgcgcctctg cctggatgag gcccbctcct gggacbgttt: cgccctctcg ctgctggctg tggctctggg ccbgggtgbg cccatgctgc nbcacctctg tggctgggac ctctggtact gcttccacct gtgcctggcc tggcttccct ggcgggggcg gcaaagtggg cgagatgagg atgccctgcc ctacgatgcc btcgtggtcb Ccgacaaaac gcagagcgca gtggcagact gggtgtacaa cgagcttcgg gggcagctgg aggagbgccg tgggcgcbgg gcacbccgcc tgtgcctgga ggaacgcgac tggctgcctg gcaaaacccl: ctttgagaac ctgtgggcct cggtcbatgg cagccgcaag acgctgtttg tgctggccca cacggaccgg gtcagtggtc tcttgcgcgc cagcttccbg ctggcccagc agcgcctgct ggaggaccgc aaggacgtcg tggtgctggt gatcctgagc ccbgacggcc gccgctcccg ctatgtgcgg ctgcgccagc gcctctgccg ccagagtgtc ctcctctggc cccaccagcc cagtggtcag cgcagcttct gggcccagcb gggcatggcc ctgaccaggg acaaccacca cttctataac cggaacttct gccagggacc cacggccgaa tagccgtgag ccggaatcct gcacggtgcc acctccacac tcacctcacc tctgcctgcc tggtctgacc ctcccctgcf; cgcctccctc accccacacc Cgacacagag caggcactca ataaatgcta ccgaaggc or transcript variant B of the following sequence tggtgaactg caactggctg ttcctgaagt ctgtgcccca cttctccatg gcagcacccc gtggcaatgt caccagcctt tccttgtcct ccaaccgcat ccaccacctc catgaCtctg actttgccca cctgcccagc ctgcggcatc tcaacctcaa gtggaactgc ccgccggttg gcctcagccc catgcacttc ccctgccaca tgaccatcga gcccagcacc ttcctggctg tgcccaccct ggaagagcta aacctgagct acaacaacat catgactgtg cctgcgctgc ccaaatccct catatccctg tccctcagcc ataccaacat cctgatgcta gactctgcca gcctcgccgg cctgcatgcc ctgcgcctcc tattcatgga cggcaactgt tattacaaga acccctgcag gcaggcactg gagcjtggccc cgggtgccct ccttggcctg ggcaacctca cccacctgtc actcaagtac aacaacctca ctgtggtgcc ccgcaacctg ccttccagcc tggagtabct gctgttgtcc tacaaccgca tcgtcaaact ggcgcctgag gacctggcca atctgaccgc cctgcgtgtg ctcgaCgtgg gcggaaattg ccgccgctgc gaccacgctc ccaacccctg catggagtgc cctcgtcact tcccccagct acatcccgat accttcagcc acctgagccg tcttgaaggc ctggtgttga aggacagttc tctctcctgg ctgaatgcca gttggttccg tgggctggga aacctccgag tgctggacct gagtgagaac ttcctctaca aatgcatcac taaaaccaag gccttccagg gcctaacaca gctgcgcaag cttaacctgt ccttcaatta ccaaaagagg gtgtcctblig cccacctgtc tctggcccct tccttcggga gcctggtcgc cctgaaggag ctggcicatgc acggcatctt cttccgctca ctcgatgaga ccacgctccg gccactggcc cgcctgccca tgctccagac tctgcgtctg cagatgaact tcatcaacca ggcccagctc ggcatcttoa gggccttccc tggcctgcgc tacgtggacc tgtcggacaa ccgcatcagc ggagcttcgg agctgacagc caccatgggg gaggcagatg gaggggagaa ggtctggctg cagcctgggg accttgctcc ggccccagtg gacactccca gctctgaaga cttcaggccc aactgcagca ccctcaactt caccttggat ctgtcacgga acaacctggt gaccgtgcag ccggagatgt ttgcccagcb cbcgcaccbg cagtgcctgc gcctgagcca caactgcatc tcgcag-gcag tcaatggctc ccagbtcctg ccgctgaccg gtctgcaggt gctagacctg tcccacaatia agctggacct ctaccacgag cactcatbca cggagctacc acgacCggag gccctggacc tcagctacaa cagccagccc tttggcatgc sgggcgtggg ccacaacttc agcttcgtgg ctcacctgcg caccctgcgc cacctcagcc tggcccacaa caacatccac agccaagtgt cccagcagct ctgcagtacg tcgctgcggg ccctggactt cagcggcaat gcacfcgggcc atatgtgggc cgagggagac ctctatctgc acttcttcca aggcctgagc ggtttgatct ggctggactt gtcccagaac cgcctgcaca ccctcctgcc ccaaaccctg cgcaaccbcc ccaagagcct acaggtgctg cgtctccgcg acaattacct ggccttcttt aagtggtgga gcctccactt cctgcccaaa ctggaagtcc tcgacccggc aggaaaccag ctgaaggccc tgaccaatgg cagcctgcct gctggcaccc ggctccggag gctggatgtc agctgcaaca gcatcagctt cgtggccccc ggcttctttt ccaaggccaa ggagctgcga gngcCcaadc ttagcgccaa cgccctcaag acagtggacc actcct;ggtt tgggcccctg gcgagtgccc tgcaaatact agatgtaagc gccaaccctc tgcactgcgc ctgtggggcg gcctttatgg acttcctgct ggaggtgcag gctgccgtgc ccggtctgcc cagccgggtg aagtgtggca gbccgggcca gctccagggc ctcagcatct ttgcacagga cctgcgcctc tgcctggatg aggccctctc ctgggactgt Ctcgccctct cgctgctggc tgtggctctg ggcct:gggtg tgcccatgct gcatcacctc tgtggctggg acctctggta ctgcttccac cUgtgccCgg cctggcttcc ctggcggggg cggcaaagtg ggcgagatga ggatgccctg ccctacgatg ccttcgcggt cttcgacaaa acgcagagcg cagtggcaga ctgggtgtac aacgagcttc gggggcagct ggaggagCgc cgtgggcgct gggcactccg cctgtgcctg gaggaacgcg acCggctgcc tggcaaaacc ctctttgaga acctgtgggc ctcggtctat ggcagccgca agacgctgtt bgtgctggcc cacacggacc gggtcngtgg tctcttgcgc gccagcttcc tgctggccca gcagcgcctg ctggaggacc gcaaggacgt'cgtggtgctg gLgatcctga gccctgacgg ccgccgctcc cgctatgtgc ggctgcgcca gcgcctctgc cgccagagtg tcctcctctg gccccaccag cccagtggtc agcgcagctt ctgggcccag ccgggcatgg ccctgaccag ggacaaccac cacttctata accggaactt ctgccaggga cccacggccg aatagccgtg agccggaatc ctgcacggtg ccacctccac actcacctca cctctcjcct;g cctggtctga ccctcccctg ctcgcctccc tcaccccaca cctgacacag agcaggcact castaaatgc taccgaaggc and is capable of specifically binding to non-methylatod CpG motifs. 16. Method for purifying body fluids from prokaryotic DNA as claimed in claim 5, wherein the separation takes place extracorporally under sterile conditions. 1:7. A kit containing at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs for enriching prokaryotic DNA by means of a method as claimed in one of the claims 1,2 or 4 to 15. 18. A test kit containing at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs and has one or more sets of specific primers for detecting prokaryotic DNA by a method as claimed in claim 15. A method is described for enriching procaryotic DNA, said method including the steps of contacting at least one procaryotic DNA with at least one protein or polypeptide which is capable of specifically binding to non-methylated CpG motifs, and separating the protein/polypeptide-DNA complex. Moreover, the application relates to a kit for carrying out said method.

Documents:

00370-kolnp-2005 abstract.pdf

00370-kolnp-2005 assignment.pdf

00370-kolnp-2005 claims.pdf

00370-kolnp-2005 correspondence.pdf

00370-kolnp-2005 correspondence_1.1.pdf

00370-kolnp-2005 correspondence_1.2.pdf

00370-kolnp-2005 correspondence_1.3.pdf

00370-kolnp-2005 correspondence_1.4.pdf

00370-kolnp-2005 description(complete).pdf

00370-kolnp-2005 drawings.pdf

00370-kolnp-2005 form-1.pdf

00370-kolnp-2005 form-18.pdf

00370-kolnp-2005 form-3.pdf

00370-kolnp-2005 form-3_1.1.pdf

00370-kolnp-2005 form-3_1.2.pdf

00370-kolnp-2005 form-5.pdf

00370-kolnp-2005 g.p.a.pdf

00370-kolnp-2005 international publication.pdf

00370-kolnp-2005 international search authority report.pdf

00370-kolnp-2005 others document.pdf

00370-kolnp-2005 pct others.pdf

370-KOLNP-2005-(13-01-2012)-FORM-27.pdf

370-KOLNP-2005-CORRESPONDENCE 1.5.pdf

370-KOLNP-2005-CORRESPONDENCE.pdf

370-KOLNP-2005-FORM 27 1.1.pdf

370-KOLNP-2005-FORM 27.pdf

370-kolnp-2005-granted-abstract.pdf

370-kolnp-2005-granted-assignment.pdf

370-kolnp-2005-granted-claims.pdf

370-kolnp-2005-granted-description (complete).pdf

370-kolnp-2005-granted-drawings.pdf

370-kolnp-2005-granted-form 1.pdf

370-kolnp-2005-granted-form 18.pdf

370-kolnp-2005-granted-form 3.pdf

370-kolnp-2005-granted-form 5.pdf

370-kolnp-2005-granted-gpa.pdf

370-kolnp-2005-granted-letter patent.pdf

370-kolnp-2005-granted-reply to examination report.pdf

370-kolnp-2005-granted-specification.pdf