DE10236711A1 - Typing genes that contain polymorphic microsatellite loci, useful for identifying predisposition to disease, by amplification and determining length of amplicons - Google Patents

Abstract

Typing (M1) a gene (I) that has one or more polymorphic microsatellite loci (PML) comprises: (a) PCR amplification of at least one DNA region of (I) that includes PML, using as template a DNA sample containing at least one segment of (I); and (b) determining the length of the resulting amplicon(s). Independent claims are also included for: (1) determining (M2) microsatellite markers (MM) for predisposition to a disease, associated with a gene that includes one or more PML; and (2) prediagnosis (M3) of diseases associated with gene that include PML.

Description

The present invention relates to a typing procedure for Genes that have at least one microsatellite locus, a method for the determination of markers in genes which contain microsatellite loci with respect to the predisposition of diseases as well as a method for the pre-diagnosis of diseases, that are associated with genes that have one or more microsatellite loci exhibit.

Genetic analyzes are mostly based on DNA variants targeted within populations or families. microsatellite are common Candidates for such investigations. However, it was believed to be on average about every 10 to 50 kbp in DNA and rarely within genes occur (see e.g. Schlötterer 2000).

With many genes related to diseases associated, no or only a few polymorphisms are known and these usually do not allow fast (i.e. efficient) typing. An example of this are those known as transmissible spongiform encephalopathies (TSE) summarized degenerative diseases of the nervous system in humans and animals, by an unusual Pathogen type are caused. Pathogen of such encephalopathies, For example, scrapie (sheep, disease known for over 250 years), bovine Spongiform encephalopathy (BSE; cattle), Creutzfeldt-Jakob disease (CJD; human) or related Eigen essentially do not contain any nucleic acids (neither DNA or RNA), but rather have an infectious character due to their protein component. this The pathogen type was called "Prion" (proteinaceous infectious particles, Prusiner 1982).

Characteristic of TSE diseases is an incubation period of several months to a few years with subsequent clinical symptoms, e.g. signs of restlessness, movement disorders, like For example, a trotter-like walk on the front legs of the sheep. in the Cases of scrapie occur over it furthermore sensitivity disorders (stronger Itching, which leads to damage to the fleece due to abrasion). The physical Condition of infected animals or of CJD patients worsened dramatically until after a few weeks it is no longer in the Are able to ingest food.

TSE diseases can be transmitted horizontally from animal to animal. Injection of an extract from the brain of a diseased animal into a healthy animal leads to infection with subsequent manifestation of the typical clinical symptoms. In the case of scrapie, the fact that nucleic acids can have no essential significance for the infection was demonstrated by experiments with scrapie brain extracts which remained infectious, even though they had previously been treated with UV light and with short-wave ionizing radiation. This regularly damages or destroys nucleic acids (Prusiner 2000). Based on these and similar experiments, other hypotheses regarding the nature of the pathogen of TSE have been pushed into the background, e.g. the virino hypothesis (Bruce and Dickinson 1987), which states that the actual pathogen is a nucleic acid that is only included by the PrP ^Sc . This also applies to the other assumption (e.g. Dron and Manuelidis 1996) that conventional viruses play a role or that an altered gene expression may be the cause.

The gene coding for the Prior protein (PrP gene) was found in the genome of all mammals examined. Its product, the cellular prion protein (PrP ^C ), is expressed especially in nerve cells, but also in cells of the placenta. So-called. "Knockout" mice, in which the PrP gene was genetically eliminated or switched off, showed no phenotypic finding after injection of infectious material (Büeler et al. 1992, 1993). The function of the PrP (e.g. Cu transport, signal transmission) has not yet been finally clarified.

TSE is probably transmitted by an isoform of the normal cell's own PrP ^C , which is referred to as PrP ^Sc (Prusiner 1998). The two forms differ in their conformation, i.e. in the way they are spatially folded. Fourier tiansformation infrared spectroscopy with highly purified PrP ^C indicated a high proportion of α-helix, whereas PrP ^Sc , i.e. the infectious form, has a large proportion of β-sheet structure (Pan et al. 1993, Caughey et al. 1991, Gasset et al. 1993). The PrP ^Sc can multiply in the host animal by catalyzing the conformational reversal from PrP ^C to PrP ^Sc and dissolving a cascade with a domino effect. This results in PrP ^Sc capable of agglomeration, which cannot be broken down by proteinase K (resistance to proteases). As a result, amyloid-like deposits form. The pathogenicity of the pathogens is due to the disturbance and impairment of the nerve tissue.

Overall, considerable efforts have been made in the prior art to gain knowledge about the structure and function of the PrP gene or PrP protein in animals and humans. For example, the PrP gene of sheep was developed in 1998 by Lee et al. sequenced. The 31412 bp sequence is stored in GenBank under the accession number U67922. The sheep's PrP gene ( 2 ) contains 3 exons with lengths of 52, 98 and 4 028 bp and 2 introns (2 421 and 14 031 bp). In addition, the database entry also includes a region of 5,665 bp 5 'wards of exon 1 and a 3' region of 5117 bp.

The 5'-UTR of the mRNA encoded by the ovine PrP gene is encoded by exons 1, 2 and 3 and consists of a total of 160 bp ( Fig. 3 ). The 771 bp DNA section that is translated (Open Reading Frame, ORF) is located in exon 3 and encodes 256 amino acids. Exon 3 also encodes the sequence of the 3'-UTR. The 3'-UTR of the ovine PrP mRNA with 3246 bp is much longer than the z. B of humans and mice (Lee et al. 1998) because they contain repetitive elements a BovB (LINE, Long Intenpened Element), a Bov tA2 / -tA3 (SINE, Short Intenpened Element) and the Mariner element (Lee et al. 1998).

The protein PrP in sheep consists of 256 amino acids, including the 24 amino acid signal peptide. The 5 repetitions of an 8-amino acid motif (octapeptide) from position 54 ( 4 ).

In the prior art at least 11 polymorphic positions in the ovine PrP gene known (Tab. 1). at all animals examined were always only one deviation found of the wild type, d. h a mutated position Since the amino acid glycine substituted at position 171 with both arginine and histidine there are a total of 12 different allelic variants of the ovine PrP gene.

The transmission of TSE succeeds only if there is homology between the donor's PrP and the PrP of the host is present, that is, the amino acid sequences have a minimum match own (Wilkens 1997). In this context one speaks in the case of transmissions between species of "species barrier" and in transmissions within a species of "polymorphism barrier". The genetic predisposition so spit on the broadcast the disease and its pathogenesis play an essential role (see also Manson et al. 2000). pronounced Effects of DNA variants within the PrP gene have been observed in humans in Gerstmann-Sträussler-Scheinker syndrome (Hsiao et al. 1989) and the hereditary Creutzfeld-Jakob disease (Palmer et al. 1991).

Three of the 11 polymorphic positions in sheep PrP (codon 136: valine or alanine, codon 154: histidine or Arginine and Codon 171: glutamine or histidine or arginine) were when examining the associations with the scrapie incubation period included (Belt et al. 1995, Hunter et al. 1996). For the description of the Haplotypes are a combination of 3 letters (e.g. VRQ: valine (V) at position 136, arginine (R) at position 154 and Glutamine (Q) at position 171). Since only one position at a time compared to the Wild type when mutated was found, a total of 5 haplotypes occur with the following names: VRQ, ARQ (wild type), ARH, AHQ and ARR.

Due to the genotyping of numerous animals from various breeds suffering from scrapie in different countries it is assumed that the haplotypes VRQ and ARQ with a increased responsiveness across from Scrapie go hand in hand. In contrast, animals with the PrP genotype ARR / ARR also showed Except for one case in Japan, no signs of scrapie disease. The rarer haplotypes AHQ and ARH are considered one in terms of scrapie susceptibility in the middle area classified.

By combining the 5 above Maintains haplotypes you have 15 different genotypes. The Scottish Scrapie Information Group "(Dawson et al. 1998) shared these 15 genotypes taking into account the breed into 5 different risk groups (R1 to R5) (Tab. 2).

The frequencies of the ORF variants In the PrP gene in 15 breeds, Drögemüller et al. (2001) The observed variants of the PrP gene were highly race-specific. So came z. B. in the Texel breed with the exception of AHQ / AHQ all 15 genotypes before, during only two or three genotypes were observed in some breeds In the breeds German Whitehead, Deutsches Schwarzkopf and Bleu du Maine had more than 50% of the Animals of the ARR / ARR genotype. In many breeds, such as Merinoland and Gotland, this genotype was not represented in those breeds relatively common Genotype ARQ / AHQ was in the breeds Deutsches Weißkopf, Deutsches Not to watch Schwarzkopf and Bleu du Maine.

While in sheep, at least, there is evidence for a connection between disease incidence and ORF polymorphisms could be found, but these do not fully explain the Scrapie distribution and breed-specific differences (O'Doherty et al. 2000). On the other hand, so far in the case of BSE (cattle) none (Hunter et al. 1994) or only minor ones (Neibergs et al. 1994) Relationships between PrP polymorphisms and the BSE incidence. There were no amino acid substitutions found in bovine PrP, and a silent mutation and a variable Repeat motif that for an octapeptide encoded in the PrP are the only known so far variabilities in the bovine PrP gene.

As stated earlier, the genotype is for the Infection risk with TSE is paramount. Hence the need for the genotype in animal and Man early To be determined in order to assess the risk of infection In the case of animal breeding educates about it In addition, the division of the genotypes into risk classes (Tab. 2) base for Breeding programs. It is said for animal breeding with gradual elimination of PrP alleles, associated with very high scrapie susceptibility to animals are bred with the resistant genotype ARR / ARR (Drögemüller and Distl 2001).

With the investigation of the genetic predisposition of the disease risk for sheep with certain genotypes or haplotypes of the PrP gene located on chromosome 13, a possibility for breeding measures, such as selection decisions, was created. Three closely coupled polymorphisms in codons 136, 154 and 171 with 5 known Haplotype forms (VRQ, ARQ, AHQ, ARH, ARR) show a close relationship to the disease rate (incubation period) with scrapie (Dawson et al. 1998). However, the examination in the PrP gene at the corresponding positions makes it complex sequencing required, the costs in 2001 in Germany were about 50 DM per animal.

The genotyping is carried out accordingly in animals and humans in the prior art by sequencing Process that is relatively expensive and time consuming. Task of It is now the present invention to provide such methods make the genotyping easier, faster and, in particular, cheaper allow in humans and animals. Furthermore, it is the task of the present Invention to provide methods that make it possible to deal with diseases, e.g. TSE, related Genes markers for a susceptibility for such Find diseases and these for quick and inexpensive prediagnostics use.

These tasks are carried out by the in the claims featured embodiments solved the present invention.

• (a) Amplifizieren mindestens eines DNA-Bereichs des zu typisierenden Gens durch PCR mit entsprechend der Größe des DNA Bereichs ausgewählten Primern, wobei der DNA-Bereich mindestens einen polymorphen Mikrosatellitenlocus enthält und als Matrize eine Probe des Individuums dient, enthaltend eine DNA, die mindestens einen Abschnitt des Gens mit dem/den polymorphen Mikrosatellitenlocus/loci umaßt, und
• (b) Ermitteln der Länge des Amplifikats/der Amplifikate von Schritt (a).

In particular, from one aspect of the present invention, there is provided a method of typing a gene having one or more polymorphic microsatellite loci in an individual, comprising the steps of:

• (a) Amplification of at least one DNA region of the gene to be typed by PCR with primers selected according to the size of the DNA region, the DNA region containing at least one polymorphic microsatellite locus and serving as a template a sample of the individual containing a DNA which contains at least encompasses a portion of the gene with the polymorphic microsatellite locus / loci, and
• (b) determining the length of the amplificate (s) from step (a).

The genotyping method according to the invention is based on the fact that in the genome of eukaryotes there are sections occur that are comparatively short and consist of repeated sequence motifs consist. The number of repetitions (also called narrow "repeats" of the motif can be "microsatellites" differentiate designated sections from individual to individual. With the number of repetitions changes therefore also the length of the respective microsatellite locus. Are with a certain species the number of sequence motif repeats and thus the length of the Microsatellite locus different between individuals or races, the microsatellite locus is called "polymorphic". So therefore To use microsatellite loci for typing individual genes, therefore it is initially on the applicability of the typing method according to the invention required that the to be typed, i.e. on belonging to a group with respect to a specific property to be analyzed for at least one gene Microsatellite locus, advantageously several microsatellite loci which has a length between different Differentiates / distinguishes individuals of a species, i.e. polymorphous is / are.

In the first step of the genotyping method according to the invention becomes the length and thus indirectly the number of repetitions of the sequence motif of the respective polymorphic microsatellite locus determined. For this according to the invention for everyone Microsatellite locus a section comprising this locus or Region of the DNA containing the gene to be typed, which the microsatellite locus to be analyzed, amplified by PCR, i.e. amplified.

Of course, one of them Sample of each individual to go out with a corresponding Contains DNA, the at least a section of the gene to be typed with the or the polymorphic microsatellite locus or loci.

The amplification takes place according to the invention by PCR (“polymerase chain reaction, polymerase chain reaction), whereby according to the each to be reproduced DNA section or area selected Primer (short DNA oligonucleotides used as starter molecules) become. The technique of PCR is well known to a person skilled in the art and described, for example, in Griffin and Griffin 2001, their disclosure content in this regard full is included in the present invention. With this approach the sample of the individual already presented above thus serves as a template, specifically the DNA it contains. The amplification by means of PCR this method characteristic advantages, especially a high Speed and the fact that even the smallest amounts of Sample DNA needed be, ultimately, a single section of the gene containing DNA molecule sufficient.

In general, all can be used as sample sources DNA-containing materials of the respective individual are used. These include e.g. blood, serum, secretions such as sweat, ejaculate etc., synovial fluid, Amniotic fluid, excrement, cerebrospinal fluid, etc. It can also be single cells or fragments thereof, for example individual skin cells, hair root cells, that are preferably on torn or fallen hair, etc. are used.

The expression "gene which has one or more microsatellite loci" means that the relevant section of the genome which contains the gene to be typed contains at least one microsatellite locus, this section includes in particular exon and intron regions of the gene and regions which are connected, in particular inherited, 5 'and 3' regions, for example those regions which are involved in the regulation of the gene in question.

The size of the amplified section of DNA according to the invention is not particularly restricted. For microsatellite positions are lengths from about 50 to about 500 bp, especially from about 50 to about 300 Bp, preferred.

In step (b) of the genotyping method according to the invention becomes the length of the amplificate or amplificates obtained in step (a). According to a preferred embodiment advantageously electrophoresis, i.e. the separation of the Amplificates carried out with the help of an electric field. As Separation medium is preferably a gel, in particular agarose or polyacrylamide gels, the production of which is familiar to a person skilled in the art; see. For example, Maniatis 2001. Particularly preferred is a length the amplificates from about 50 to about 300 bp, their separation in gels, the a separation of the fragments down to a few or even Allow 1 bp. Such gels are also used, for example, in the sequencing of DNA molecules. The use of automatic laser fluorescence DNA is particularly advantageous sequencers (e.g. A.L.F. from Pharmacia) with corresponding gels (e.g. Hydrolink), the use of internal and external standard fragments, the commercially available are, or in a simple manner by means of PCR starting from a known Template DNA, such as λ DNA, can be made is advantageous as a comparison.

Experience has shown that the number of repetitions of the respective sequence motif is a rough indicator of the degree of polymorphism of the microsatellite locus. Of course, microsatellite loci are generally all the more informative, that is, the more individual microsocial loci are available, the more the number of sequence motif repetitions in the microsatellite locus fluctuates between the individual, the more the number of subspecies can be categorized. Accordingly, it is preferred according to the invention if the polymorphic microsatellite locus / loci has a sequence motif which is repeated at least three times, more preferably at least four times, particularly preferably at least five times. Polymorphic microsatellite loci which are selected from the following consensus sequences can be mentioned as examples: (ATA) ₇ , (GA) ₇ , (GT) ₂₈ , (AGG) ₄ , (AC) ₅ , (TTTGT) ₅ , (TA) ₇ , (TC) ₅ , (TAAA) ₁₂ , (ATTTT) ₁₂ , (TTTC) ₁₈ , (CA) ₁₈ , (CAGTT) ₄ , (AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (TTCAG) ₄ , (ACTGA) ₃ , (ACTGA) ₄ , (CA) ₁₂ , (ATC) ₄ , (T) ₂₅ , (TTG) _5, (T) _{2 9} , (CAA) ₃ , (TTCAG) ₃ , (AGTG) ₄ , (CAC) ₅ , (AAAACA) ₄ , (ACC) ₄ , (ATCAG) ₅ , (AC) ₃ , (CTG) ₅ , (GTT) ₅ , (T) ₁₅ , (CAA) ₄ and (AC) ₄ .

Basically, the typing method according to the invention can be perform on all genes that have at least one polymorphic microsatellite locus. Whose or their sequence and position in the gene to be typed can on the one hand already known. On the other hand, the polymorphic (s) Microsatellite locus / loci in a preferred embodiment of the method according to the invention be determined.

The identification of the polymorphic microsatellite locus / loci is preferably carried out as follows:
In a first step (A), the gene to be typed is screened for microsatellite loci, ie screened. This generally requires a genomic sequence of the gene in question which has at least one microsatellite locus. Genomic sequence information relating to a large number of genes is known in the prior art and is in particular stored in the databases of GenBank and the EMBL under corresponding access numbers. For example, if only one cDNA sequence is available or only EST sequences, it is of course possible for a person skilled in the art to obtain the required genomic sequence information using the conventional molecular biological methods; see also Maniatis et al. 2001. The genomic sequence is preferably screened for microsatellite loci using appropriately designed computer programs. As examples of suitable software, the programs can be found in the program package EMBOSS (from HGMP-RC, London, Sputnik (Abijian 1994) and software RepeatMasker (Smit and Green, http :: //ftp.genome.washington.edu/cgibin/repeatmasker) On the other hand, even if there is very little or no sequence information at all for a gene to be typed from a certain species, it is possible to also type this gene or to determine polymorphic microsatellite loci located therein, if in another species the gene sequence is known to a sufficient extent and microsatellite loci located therein have also been identified. In this case, with the proviso that there is sufficient sequence homology with regard to the gene to be typed between the species, for example that of the species, sufficient sequence information is available is used, primer also used in the course of typing the unknown gene of the other S Specification to be performed amplification of the corresponding DNA sections can be used. In this case, it is therefore also possible to determine polymorphic microsatellite loci in a gene with only an incomplete or no (genomic) sequence at all, without screening this gene for microsatellite loci per se.

In a (further) step (B) analogous to the above step (a) of the invention Genotyping method the microsatellite loci obtained during the screening of the gene corresponding DNA areas (one DNA section or area per microsatellite locus obtained) in a plurality of individuals of the organism containing the gene to be typified amplified by PCR, ie amplified. With regard to the amplification, the statements made above for step (a) of the above typing method also apply.

In the next step (C) - analog to step (b) of the typing method according to the invention - the length of the Amplificate or the amplificates determined in each individual were obtained for the length determination the rest of the amplificates apply the above statements in terms of Step (6) of the genotyping method according to the invention accordingly additional to determine the length of the amplificates obtained for each individual or alternatively according to the invention, a Sequencing of the respective amplificate or a section of which, however, contain at least the microsatellite locus itself must be performed. Suitable sequencing methods are well known to a person skilled in the art and can in particular by means of automated sequencing devices (e.g. A.L.F. by Pharmacia).

Finally, the lengths of the Amplificates between the individuals or the nucleotide sequences obtained compared with each other So in the last step (D) the or the microsatellite locus / loci determined, its or its amplificate (s) has / have a different length between the individuals or its nucleotide sequence (s) differ between individuals is / are. These microsatellite loci differ between individuals (in terms of which therefore have different alleles) are polymorphic. As already explained above, using the typing method according to the invention the most diverse genes are typed prerequisite just that the gene to be typed at least one polymorphic microsatellite locus contains. The typing method according to the invention is preferred applied to genes that directly or indirectly with the expression of a disadvantageous or advantageous feature of an organism associated are. With regard to the disadvantageous expression of a feature are here especially to mention genes associated with a disease or other important from a medical or economic point of view (e.g. in (farm) animals) Traits are associated with specific examples of such genes are PrP (BSE diseases, such as scrapie in sheep, BSE in cattle, Creutzfeld-Jakob disease in humans), CFTR (cystic fibrosis), Genes associated with metabolic disorders in humans and animals are like α-manosidosis, Pompe disease, citrullinemia and Bovine leukocytic adhesion deficiency in cattle (each a defective gene, its variants at the DNA level are distinguishable), susceptibility to stress (Malignant hyperthermia syndrome) in pigs (RYR1 gene, variant T), genes, milk proteins, hormones or transcription factors encode and so on of the organism whose respective gene is to be typed none according to the invention Limitations, because microsatellite loci can be found in all eukaryotic genes. Due to economic or scientific considerations are preferred examples of organisms and others. Man, monkey, Horse, sheep, cattle, pig, goat, mouse, rat, rabbit, guinea pig, To name dog, cat and chicken.

Due to the availability of diverse sequence information and the importance in population studies, a particularly preferred target gene of the method according to the invention is the PrP gene already described above, which is associated with TSE diseases which occur in humans and other mammalian species. In particular, the genotyping method according to the invention is applied to the human PrP gene, the sequence motif (s) of the polymorphic microsatellite locus / loci being selected from the following consensus sequences: (ATA) ₇ , (GA) ₇ , ( GT) ₂₈ , (AGG) ₄ , (AC) ₅ , (TTTGT) ₅ , (TA) ₇ , (TC) ₅ , (TAAA) ₁₂ , (ATTTT) ₁₂ , (TTTC) ₁₈ and (CA) ₁₈ .

The consensus sequences (ATA) ₇ , (GA) ₇ , (GT) ₂₈ , (TAAA) ₁₂ , (ATTTT) ₁₂ and (CA) ₁₈ are particularly preferred.

Furthermore, it is regarding the Human PrP gene preferred when the amplified DNA region (s) in step (a) of the genotyping method according to the invention nucleotides 3621 to 3641, 25415 to 25428, 39450 to 39505, 50315 to 50326, 58346 to 58385, 72392 to 72416, 78300 to 78313, 92615 to 92624, 108429 to 108476, 116907 to 116966, 125528 to 125599 and / or 147980 to 148015 according to GenBank accession number AL133396 comprises / comprise. In particular, DNA regions are for genotyping according to the invention for amplification according to step (a) preferred which the nucleotides 3621 to 3641, 25415 to 25428, 39450 to 39505, 108429 to 108476, 116907 to 116966 and / or 147980 to 148015 according to GenBank Include access number AL133396.

Preferred sequence motifs of the polymorphic microsatellite locus / loci of the bovine PrP gene have the following consensus sequences: (CAGTT) ₄ , (TC) ₅ , (AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (TTCAG) ₄ , (ACTGA) ₃ , (AGTGA) ₄ , (CA) ₁₂ , (ATC) ₄ , (T) ₂₅ , (TTG) ₅ , (T) ₂₉ , (CAA) ₃ , (TTCAG) ₃ , (AGTG) ₄ , (CAC) ₅ , (AAAACA) ₄ , (ACC) ₄ , (ATCAG) ₅ and (AC) ₃ .

The consensus sequences (AGC) ₅ , (TTTGT) ₄ , (CA) ₁₂ , (T) ₂₅ , (T) ₂₉ , (TTCAG) ₃ , (AAAACA) ₄ and (ATCAG) _{5 are} particularly preferred.

In the bovine PrP gene, DNA regions which comprise the nucleotides are preferably amplified 444 to 463, 4121 to 4130, 7615 to 7629, 19808 to 19817, 25288 to 25307, 30633 to 30652, 32320 to 32339, 37891 to 37910, 39943 to 39957, 44220 to 44239, 44507 to 44530, 46259 to 46278, 48409 to 48420, 50471 to 50495, 53219 to 53233, 54990 to 55018, 60452 to 60460, 62703 to 62717, 64685 to 64700, 66178 to 66192, 68762 to 68785, 68926 to 68937, 72474 to 72498 and / or 74333 to 74340 according to GenBank access number Include AJ298878. More preferably, the amplified DNA regions of the bovine PrP gene comprise nucleotides 7615 to 7629, 25288 to 25307, 44507 to 44530, 50471 to 50495, 54990 to 55018, 62703 to 62717, 68762 to 68785 and / or 72474 to 72498 according to GenBank accession number AJ298878.

According to a further preferred embodiment, the gene to be typed is the PrP gene of the sheep. The polymorphic microsatellite loci preferably have the following sequence motifs (consensus sequences): (CAGTT) ₄ , (TC) ₅ , (AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (ACTGA) ₃ , ( ACTGA) ₄ , (CA) ₁₂ , (CTG) ₅ , (ATC) ₄ , (GTT) ₅ , (TTG) ₅ , (T) ₁₅ , (CAA) ₃ , (CAA) ₄ , (TTCAG) ₃ , ( AGTG) ₅ , (CAC) ₅ , (ACC) ₄ and (AC) ₄ . Microsatellite loci in the sheep's PrP gene with the consensus sequences (GA) ₅ , (TTTGT) ₄ , (CA) ₁₂ , (GTT) ₅ , (T) ₁₅ and (CAA) ₄ are particularly preferred.

In the typing according to the invention of the sheep PrP gene include those amplified according to step (a) above DNA regions the nucleotides 301 to 320, 590 to 613, 1033 to 1047, 2477 to 2496, 4651 to 4662, 6627 to 6641, 9433 to 9447.11277 to 11291.16748 to 16756, 18100 to 18119, 19372 to 19386, 21383 to 21398, 22853 to 22867, 25422 to 25433, 25580 to 25591 and / or 30168 to 30175 according to GenBank Accession number U67922, as well as seven other genomic DNA areas, the 5 'of the sequence disclosed in U57922 are located and for which none GenBank entry exists. DNA regions of the sheep PrP gene are particularly preferably amplified include nucleotides 590 to 613, 6627 to 6641, 11277 to 11291 and / or 25422 to 25433 according to GenBank accession number U67922, as well as seven other genomic DNA regions, the 5 'of are located in U67922 sequence and for which no GenBank entry exists.

From the point of view of genotyping is thus the use according to the invention provided by microsatellites for typing genes. there are for the first time in particular in the PrP gene of humans, cattle and sheep Microsatellites deployed between the individuals of the individual species, i.e. are polymorphic Microsatellite loci several different alleles exist.

From another point of view The following invention uses the genotyping method described above used in a process that is suitable for microsatellites Markers for the predisposition of an individual regarding to determine a disease, the disease with a gene is associated with one or more polymorphic microsatellite loci having.

In a first step (i) this The corresponding gene identification procedure is used in a majority of individuals according to the above typing method described typed. Regarding special embodiments this typing procedure is based on the above referred to in the first aspect of the invention by everyone the majority of individuals know, for example, that it is the Disease, for example diseases mentioned above, or Not. In addition or alternatively, for example from examinations known in the prior art the respective disease with other genetic markers, e.g. ORF polymorphisms, restriction fragment length polymorphisms, etc., from be known to every single individual of the plurality of individuals, that this each individual has a certain degree of predisposition to the disease having. A specific example of such a relationship between known genetic markers and the degree of predisposition a disease can be the ORF polymorphism described above in the ovine PrP gene (see Tables 1 and 2). Preferably in step (i) at least 100, particularly preferably 500 or genotyped more individuals.

In a further step (ii) of the identification method according to the invention for microsatellite markers become the genotype frequencies for each polymorphic microsatellite locus in the individuals, of which is known that they are ill or not or a certain degree of predisposition for the Have disease, determined.

Finally, in the last step (iii) it is determined in which of the polymorphic microsatellite loci there is the greatest statistical significance for the characteristic “sick” or “not sick” or for the known specific degree of predisposition on the basis of the genotype frequencies determined in the previous step (ii) , If, for example, an expression of the polymorphic microsatellite locus is found exclusively in healthy individuals, ie individuals who do not suffer from the disease in question, and the frequency of this genotype, ie this allele, is increased or particularly high in non-diseased individuals, the polymorphic microsatellite locus in question is used as a marker be suitable for a lack of predisposition to the disease and thus be selected. In contrast, a variant, ie an allele, of a microsatellite locus that is found exclusively or frequently in diseased individuals is selected as a microsatellite marker for an increased predisposition to the disease in question. Between these extremes, ie from Finally, in the case of healthy manifestations or those occurring exclusively in diseased individuals, intermediate stages are of course also possible, which nevertheless correlate with a certain degree of predisposition. As already explained above, the specific degree of predisposition can also be related to the invention on the basis of other genetic markers which are known to correlate with a specific degree of predisposition. As an example, the ORF polymorphism known in the ovine PrP gene may be mentioned here. In the case of the ORF polymorphism in the ovine PrP gene known in the prior art, it can be assumed on the basis of the genotyping of numerous sheep of various breeds from various breeds and in different countries that the ORF alleles (ORF: narrowed “open reading frame”, open reading frame, ie variants in protein coding gene regions) VRQ and ARQ are associated with a greatly increased susceptibility to scrapie, whereas animals with the genotype ARR / ARR showed a particularly high scrapie resistance (with the exception of one observation in Japan, sheep with the Genotype ARR / ARR never found a scrapie disease.) The other ORF alleles described are arranged in terms of scrapie susceptibility (see Dawson et al. 1998, Bunter et al. 2000).

Furthermore, the above are described polymorphic microsatellite positions in the ovine PrP gene in the region of this gene (with the proviso that the ovine PrP gene has no insertions across from which has the bovine PrP gene in this area is the farthest 5 'microsatellite locus is only 29 kbp from exon 1; the microsatellite locus furthest in 3 'is even located in exon 3) and their inheritance is therefore very close to that of the ORF variants coupled. Therefore, coupling breaks will be like this seldom happen that they cannot be observed within races. Thus, outgoing from the statistically excellent verified ORF genotyping in conjunction with their coupling to the polymorphic microsatellite positions in the sheep PrP gene with certainty on an association of the microsatellite variants with the scrapie resistance or susceptibility that are in same order of magnitude how it is with the ORF variants. This is explained in the following embodiment 3 direct experimental evidence found confirmed. The example of the ovine PrP gene can be Of course to other genetic markers in other genes and other organisms transferred analogously, which has no theoretical or experimental difficulties for a person skilled in the art will prepare.

Regarding organisms and genes, in which with the aid of the inventive detection method microsatellite markers there are therefore no restrictions to be identified. Preferred organisms or genes are above in terms of The genotyping method of the present invention is detailed.

Another aspect of the present The invention relates to a method for the pre-diagnosis of diseases. The diseases are associated with a gene that at least has a polymorphic microsatellite locus. The pre-diagnosis The method of the present invention initially again comprises a typing step (1) using the genotyping method defined above carried out becomes. The gene of an individual to be examined is related to a or more microsatellite locus / loci, which is a marker for the degree of predisposition in terms of the respective disease is typed. In another Step (2) then becomes the genotype obtained with that genotype assigned predisposition level in terms of compared to the disease.

The microsatellite markers are preferred in this pre-diagnostic procedure determined using the identification procedure described above. Thus, regarding of genes, organisms, etc., from the point of view above embodiments of the present invention.

The prediagnostic method according to the invention is particularly useful for TSE prediagnostics used, especially the human PrP gene, the bovine PrP gene as well as the ovine PrP gene are preferred in prediagnostics of the human PrP gene are suitably polymorphic microsatellite loci used, which have one of the sequence motifs, the above in terms of the typing method according to the invention Preferred amplified DNA regions for TSE diagnostics are specified in humans are also above from the point of view of the typing method according to the invention specified. In the case of the bovine PrP gene, polymorphic microsatellite loci are used preferred which the above with regard to the typing method according to the invention have specified sequence motifs. Preferably amplified DNA regions include those above regarding the typing procedure positions defined by the present invention. With scrapie prediagnostics (Sheep PrP gene) are advantageously polymorphic microsatellite loci used with sequence motifs, their consensus sequences also are listed above in the typing procedure. Regarding preferred positions of such microsatellite loci in the sheep PrP gene, which suitably at first conducted Genotyping in the prediagnostic method according to the invention are amplified, is also based on the above statements on Typing methods of the present invention are referenced.

The present invention is accomplished by the figures and tables described below are explained in more detail:

1 shows the tertiary structure of the prion protein (from: Pringle: BSE, Scrapie, CJD and the Prion Protein, http://www.uni-mainz.de/~cfrosch/bc4s/prions.html), whereby in 1a the cell's own form (PrP ^C ) and in 1b the pathogenic form of the protein in scrapie (PrP ^Sc ) is shown.

In 2 the exon / intron structure of the ovine PrP gene is shown. The information used relates to GenBank entry U67922,

3 represents the structure of the transcript of the ovine PrP gene (from Geldermann et al. 2002).

4 is a representation of the ovine PrP primary sequence (Geldermann et al., 2002). The sheep's PrP protein therefore consists of 256 amino acids, including an N-terminal signal peptide of 24 amino acids. ¹⁾ denotes a 5-fold repetition (5 × R) of a motif from 8 or 9 or: 5 amino acids (PQGGGGWGQ, (PHGGGWGQ) ₃ or PHGGG) that is conserved in the PrP protein. The respective amino acid and its position of the PrP alleles ( ²⁾ ) are given (see Table 1). The darkly marked amino acid variants were used in association analyzes with scrapie susceptibility.

In 5 the microsatellite loci in the ovine and bovine Prp gene are shown schematically. Microsatellite motifs with ≥ 3 repeats (repetitions) and> 90% homology were identified with the help of the program etandem of the EMBOSS program package. Additional microsatellites were identified using the information in Lee et al. (1998) (marked ¹ ), the Sputnik program (Abajian 1994) (marked ² ) or the Repeat Masker program (marked ³ )). If a microsatellite locus was found in only one species with these measures, the homologous location in the DNA of the other species was also searched. Microsatellite loci identified using this method are marked with ⁴ ). The reference scale in the middle refers to the sequences stored in GenBank (cattle: accession number AJ298878; sheep: accession number U67922). The 5 'ends of the ovine and bovine exons 3 are aligned with one another. The dotted line in the sheep gene shows an area for which no sequence information is available in GenBank, only estimated positions of the microsatellite loci being indicated. Microsatellite loci homologous in both species are identified by the same numbers. Loci S01 to S06 and S09 in sheep were examined with primers designed for the PrP sequence in cattle. The primers for the loci in cattle R07 and R08 gave no PCR products in sheep. For further details, please refer to Table 3 described below.

6 shows an example of a comparison of allele DNA sequences of selected polymorphic microsatellite loci. The respective sequence from GenBank is given as reference (R) in the first line. The different residues are marked in the allele sequences. 6A shows the comparison of allelic DNA sequences for the locus S11 in the oval PrP gene. 6B shows a corresponding comparison for a further polymorphic microsatellite locus in the PrP gene of sheep (S15). 6C shows a comparison of all DNA sequences of the locus R21 in the bovine PrP gene.

7 shows a diagram in which the number of microsatellite motifs per kbp in the PrP gene of cattle and sheep (black bars) with the corresponding values in other genes (bright bars) for different gene areas, namely exon, intron and 5 'area, as well as over the entire gene range are striking is the significantly increased density of repeat DNA in exons compared to other genes in the ovine and bovine PrP gene (p <0.05). The detailed data regarding the 7 are given in Table 6 described below.

In 8th the experimental procedure for identifying suitable microsatellite markers for TSE diagnosis is shown schematically using the example of analysis in sheep.

9 gives examples of electropherograms of the microsatellite locus S15. Lane 1 shows a homozygous, lane 2 and 3 heterozygous genotypes.

In 10 there are examples of electropherograms for the S11 microsatellite locus. Lanes 1, 2, 3 and 6 show different animals with a heterozygous genotype, lanes 4 and 5 represent homozygous genotypes.

In 11 examples of Locus S24 electropherograms are shown. Lane 1 and 2 show homozygous, lane 3 a heterozygous genotype. In lane 4 a PCR product with the length 152 occurs in combination with the allele 147, in lane 5 it can be observed together with the alleles 144 and 147.

In the 9 to 11 some examples of amplicons for the loci S15, S11 and S24 are shown as a result of the fragment length analysis of the microsatellites. All homozygous genotypes showed well-formed peaks with no slippage. In the heterozygous observations, the two allelic PCR products were clearly separated even at 2 bp distance (see 10 , Tracks 1, 3 and 6). Misleading by-products were not found on any gel.

A special feature was observed at Locus S24. A fragment of length 152 occurred in 25 merino landscapes, either in combination with 144 or 147 ( 11 , Track 4) or in 15 observations with both other alleles simultaneously ( 11 , Track 5). In order to clarify the nature of these 3 alleles, they were sequenced after previous cloning. The fragment lengths measured with the ALF were each 2 bp shorter than those determined by sequencing (Tab. 16).

12 is an alignment of the 3 sequenced S24 alleles with a section of the corresponding database entry in GenBank. The microsatellite of the database sequence consists of 4 repetitions of the CAA motif. The 147 bp long allele of sheep S102 corresponds to that except for a point mutation from A to C. Database entry. Due to this mutation, the allele 147 contains one more repetition of the motif CAA with the same length as the database sequence. The microsatellite of sheep 33 allele 144 consists of only 3 repetitions of the motif, which makes this fragment 3 bp shorter than the GenBank allele. In addition, an SNP from C to T occurs at position 57 (based on the GenBank sequence). The allele with fragment length 152 (sheep 311) is identical to allele 147 with regard to the microsatellite. However, there is a 5 bp insert between positions 29 and 30. In addition, an A occurs at position 103 instead of a T.

On the tables attached in the appendix 1 to 24 are used at the appropriate points in the present description related. In these tables, those of the present invention underlying principles and according to the following embodiments results obtained.

Tab. 1 shows the PrP gene amino acid polymorphisms found in sheep summarized (after Hunter 2000, supplemented).

Tab. 2 shows the risk classification (Risk) due to the amino acid polymorphisms in positions 136, 154 and 171 of the sheep PrP as described by the "Scrapie Information Group" (Dawson et al. 1998) was made.

Table 3A shows the data from 24 microsatellite loci with 3 or more repetitions of motifs, those found in the bovine PrP gene Tab. 3B shows the corresponding Data from the 23 microsatellite loci found in the sheep's PrP gene. 9 for the Bovine DNA-specific primer pairs were also used as sheep DNA Matrix tested to the previously unpublished area of the sheep sequence (approx. 44 kBp) could be analyzed using this procedure 7 microsatellite loci are identified in the sheep PrP gene in which both for Sheep as well for Sequence information available in GenBank was found at 13 of 16 of the loci examined in sheep have homologous microsatellite motifs found in the bovine DNA sequence. These motifs included 4-5 Repetitions, in most cases but with different sequences of mononucleotide repeats, those in introns 1 and 2 of the bovine gene and in intron 2 of the sheep gene were found to show 15 and more repetitions.

Tab. 4 shows a compilation of the results of an analysis of allelic PrP microsatellite DNA variants in the bovine (Tab. 4A) and ovine (Tab. 4B) PrP gene. Allelic variants were obtained in about 30% of the analyzed loci As in Tab 4 summarized, 4 of the 8 variable microsatellite loci showed 2 alleles in cattle, and 2 loci were highly variant (6 or 10 alleles). The data for the sheep gene in Table 4B show a locus with 2 alleles, 2 loci with 3 alleles and 3 loci with 4 and more alleles. Some of the variants were only found in one breed (9 out of 31 alleles at the riad and 2 from 22 alleles in sheep), while the other variants occur in more than one breed. The DNA sequences of allelic fragments showed differences in different positions, even throwing within a certain allele (cf. Tables 4 and 6 ). In addition to polymorphic repeats and SNPs in microsatellite motifs, variable sites (SNPs as well as insertions and deletions) were also found in the non-repetitive sequences flanking the microsatellite repeats. In 7 of 12 loci none of the sequenced alleles corresponded to the DNA sequences stored in GenBank. In 2 polymorphic loci no database sequence information available. Examples of sequenced alleles from 3 of the microsatellite loci are in 6A shown and show a variability of the fragment lengths, which result from the variable number of repetitions of the microsatellite motif. In cases with composite microsatellite sequences, the number of repetitions in each motif was independently variable ( 6B ). However, it was often found that the flanking areas of the repeat motifs were variable ( 6B and 6C ) and contained SNPs, insertions and deletions. This leads to the complex differences in some of the alleles, which is summarized in Table 4. As further shown in Table 4, the fragment lengths of the alleles were measured with the ALF DNA sequencer and compared with the number of nucleotides of the analyzed DNA sequences. In 35 of 41 cases, the fragment length analysis with the ALF device showed 1-3 nucleotides shorter values ( 1 nucleotide in 15 cases, 2 nucleotides in 16 cases and 3 nucleotides less in 4 cases) than the results obtained by sequencing. These deviations are probably due to secondary structure formation due to the repeats in the microsatellite DNA fragments, which influence the mobility of the respective species. This deviation from the actual length does not matter for the typing of genes, since the fragment length measured by electrophoresis for any given genotype is constant and therefore a characteristic value results for each allele when using the same electrophoresis system.

Tab. 5 shows those found to be polymorphic Microsatellite loci for bovine (Tab. 5A) and ovine (Tab. 5B) PrP gene together In 6 of the polymorphic microsatellite loci in cattle as well as all 6 polymorphic microsatellite loci of the sheep a frequency of the most common Alleles of <0.95 detected. Overall, a degree of heterozygosity of> 0.10 was found in 11 of the total 14 polymorphic loci found in both species were observed.

Table 6 shows comparative data for the microsatellite loci of bovine and ovine genes. Thereafter, microsatellite loci can be localized not only in the PrP gene of sheep and cattle but also in other genes of this species. On average, 1.12 microsatellite loci per 1 kBp DNA were obtained in the 8 genes analyzed. The results according to Table 6 are as described above 7 graphically darge provides.

Table 7 shows the microsatellite loci determined in the human PrP gene, of which 5 showed more than 1 allele in 18 healthy volunteers examined. The frequency of the predominant allele was <0.95. The observed degree of heterozygosity varied between 0.11 and 0.83. The PCR conditions with regard to the addition temperature and MgCl ₂ concentration and the primers used are also given.

Tab. 8 shows the number of below embodiment 3 examined sheep and their ORF genotypes are shown.

The PCR approaches are shown in Table 9 Amplification of microsatellite loci S11, S24 and S15 in the PrP gene of the Sheeps specified, Tab. 10 shows the PCR program carried out and Tab. 11 shows the sequences of the primers used.

Table 12 shows the running conditions specified in the electrophoresis, which is used to separate the microsatellite loci amplicons carried out has been.

Tables 13 to 15 contain information summarized for the sequencing of cloned PCR products, which is involved in the amplification of the microsatellite locus S24 of the PrP gene of the sheep revealed.

Table 16 shows the results of the fragment length analysis of the microsatellite locus S24 of the sheep PrP gene obtained by electrophoresis or sequencing (cf. also those described above 9 to 11 ).

The allele frequencies and frequencies the polymorphic microsatellite loci in the PrP gene area for the 8 sheep breeds examined are shown in Tab. 17. For Locus S11 the allele Fragment length 152 as the most common Allele found in race with lowest allele frequency 0.54 Suffolk and highest Frequency 0.93 in the Merinoland and Schwarzkopf breeds. Another high allele frequency was 158 in the Suffolk breed for the allele fragment length are found to be 0.45. All other frequencies to this locus were for the individual races between 0.007 (4 observations on fragment length 154 for race Merinoland) and 0.149 (26 observations on fragment length 158 for milk sheep).

Of the 5 allelic fragment lengths too Locus S11 was only fragment lengths 150 and 152 in all 8 Breeds detected The fragment length 154 was only seen in the Merinoland breed.

The counting of the allelic fragment lengths to locus S24 was carried out in a simplified form. For this locus, fragment lengths were detected which could not be clearly assigned to the locus (3-fold repeat) or which should be made accessible for interpretation by sequencing. These are the fragment length 146 (6 observations across all races) coded to 147 and the fragment length 152 (25 observations in the Merinoland breed, which only occurred in combination with 144 or 147 and was detected as the 3rd peak in 15 observations ( 11 ). This fragment length was negated as information. The analyzed fragment lengths 144 and 147 were found in all breeds, whereby fragment 144 was most frequently represented in the Dorper, Isle de France, Merinoland, Schwarzkopf and Texel breeds with frequencies between 0.61 and 0.885 and fragment 147 in the Gotland, milk sheep breeds and Suffolk was the most common allele with frequencies between 0.53 and 0.75.

The microsatellite locus S15 showed 3 allelic fragment lengths with the most common Fragment 179 in all races and frequencies between 0.57 (race Gotland) and 0.98 (Dorper breed). The fragment length 182 was in all races with low frequencies between 0.008 (1 observation for breed Suffolk) and 0.192 (33 observations on breed dairy sheep). Yet fragment 173, which was missing in 4 races, was less common with frequencies between 0.016 (2 observations on the Suffolk breed) and 0.286 (4 observations on the Gotland breed) occurred.

frequencies and frequencies for the amino acid variants of the PrP genotypes are listed in Table 18. For allelic forms Codon 136 (ORF1 label) could be used for the amino acid alanine (A) for the individual breeds found frequencies between 0.885 and 1.0 become. The amino acid Valine (V), in the literature with a high risk of scrapie susceptibility in animals with this expression specified, could only be used with low frequencies up to 0.115 (Isle of de France) are observed, being in the Gotland and dairy sheep breeds no animal appeared with this variant.

The allelic variant histidine (H) was present in very small numbers on codon 154 (ORF2 marking) (highest Frequency 0.258 in dairy sheep). This amino acid expression was missing in all breeds, except milk sheep (46 observations), Merinoland (40 observations) and Texel (1 observation). The amino acid arginine (R) was found on this Position with frequencies above 0.74 found.

At codon 171 (ORF3 marking) could for the 8 races 3 allelic forms be determined. The amino acid Histidine (H) occurred at a very low frequency with the exception of Texel breed, frequency 0.21 (30 observations). This amino acid expression could observed with one observation only in the Merinoland breed become. The characteristics Glutamine (Q) and arginine (R) were very different in the races Frequencies represent glutamine in the Gotland breed with 20 observations (100%), Arginine in the Schwarzkopf breed with 70 observations (80%).

The observed degrees of heterozygosity (DC: direct count estimate) and the undistorted heterozygosity Deep estimation values (unb: unbiased estimate) of the 3 microsatellite loci and 3 ORF loci including the mean degrees of hetrozygoty are shown in Table 19.

For the microsatellite locus S11 was the lowest observed degree of heterozygosity in the Dorper breed at 0.13 and the highest in the Suffolk breed be found to be 0.50. The microsatellite locus S24 was the lowest observed degree of heterozygosity in the breed, 0.231 Isle de France and the highest at the Suffolk breed can be recognized with 0.594. To the microsatellite locus In S15 the values were between 0.044 for the Dorper breed and 0.714 for the Gotland breed. The lowest mean observed degree of heterozygosity with 0.157 wound in the Schwarzkopf breed and the highest in the Gotland breed found at 0.571. The observation values showed in all Breeds small, insignificant deviations from the expected values.

In the lower part of tab. 19 the heterozygosity of the ORF loci is shown. The highest degree of heterozygosity at the Locus ORF1 was observed with the breed Isle de France with 0.231. The ORF2 was 0.315 for the milk sheep breed and ORF3 the highest levels of heterozygosity were found for the Texel breed 0.662. Frequently only homozygous animals occurred at the ORF loci in the individual breeds on (Gotland and milk sheep at ORF1, Dorper, Gotland Ile de France, Schwarzkopf and Suffolk on ORF2 and Gotland on ORF3).

The locus S11 is the with 5 alleles the highest polymorphic locus in the investigation. When checking the genotype frequencies the micro satellite loci on the equilibrium situation (Hardy-Weinberg equilibrium) There were no deviations in the examined breeds (Tab. 20) be determined.

At the Locus S24 with the alleles 144 and 147, the heterozygous animals were overrepresented in all breeds. The genotype 144/147 was transferred the breeds counted 23 observations (267 observed, 244 expected) too often on. This resulted however, in no breed there was a significant difference in genotype frequencies from an equilibrium situation.

The locus S15 showed 3 appearing Alleles with the rare alleles 173 and 182 no deviation from Hardy Weinberg Equilibrium (HWE) in one of the breeds.

Regarding the examination of the Genotype observations of the ORF loci could lead to the ORF1 locus (codon with the amino acid variants A (alanine) and V (valine)) did not differ from the HWE in any race are determined (Tab. 21). They only came here in the races Dorper and Texel made notable V observations, with only one Sheep of the Texel breed was recognized homozygously with V / V. At the locus ORF2 (Codon 154 with the amino acid variants H (histidine) and R (arginine) with very low H content observed in the breeds Merino country, milk sheep and Texel (1 observation) were also found no significant deviation from the HWE. An underrepresentation the heterozygote could be recognized in milk sheep.

The ORF3 locus (codon 171 with amino acid variants H, R and Q (glutamine) were significant in the Merinoland breed Deviation (P <0.05) from HWE with significant underrepresentation of type Q / R (14th Too few observations). This guy joined the Suffolk breed 6 observations (not significantly different from HWE) too often.

The rare form H (histidine) was almost exclusively represented in the Texel breed and showed no abnormality in the frequencies.

The association is shown in Tab. 22 to 24 the microsatellite loci for the scrapie risk groups (for the definition of risk groups (characteristic risk) see Tab. 2)

When analyzing variance with a model 1 (Significance and measures of certainty; see also the relevant versions in the third embodiment below) to breeds with animal numbers> 60 the following resulted. In the Merino breed, the Microsatellite loci only accounted for 7.6% of the phenotypic Variance of the Risk characteristic (Table 22) can be explained, with the Loci S11 and S15 contributed no share. In table 8 you can see that regarding the Merinoland breed risk assessment mainly was found in levels 3 and 4 (90.5% of the observations) and thus a little phenotypic Variance regarding of the examined feature showed.

For the breed milk sheep, Suffolk and Texel, on the other hand, were able to measure the model 1 with widths> 75 % are determined, with S24 with a high level of significance in all breeds Had a share in the variance of the risk characteristic.

Finally the following resulted estimates for characteristic risk from the analysis with model 1 to the considered Microsatellite loci. The analyzes of the individual breeds (Tab. 23) resulted in the Locus S24 for Animals with genotype 147/147 compared to animals with the genotype 144/144 a clear increase in Characteristic risk with the highest Differentiation in the Suffolk breed between the two homozygous types with an expected selectivity of about 2.7 in the risk difference. The locus S11 showed up a higher one Characteristic value risk in animals with the allele 150, particularly good too recognize the Texel breed. However, there were only heterozygous observations in front.

The Locus S15 points to the individual Breeds inconsistent association sizes for the different genotypes. showy was an increase in risk in the Texel breed, detectable for animals with the allele 173 occurred.

The examined ones are in Table 24 Microsatellite loci listed within ORF genotypes, with within of these classes the microsatellite loci in the sort order S24, S11, S15 by genotype. Within that with the least Disease risk associated ORF type ARR / ARR with 76 observations in 6 breeds showed all animals the same genotype combination of the 3 Microsatellite loci S24, S11, S15 with 144/144, 152/152 and 179/179. This homozygous expression At the three loci there were 188 observations in the entire data material in front with proportionately decreasing Trend up to risk level 4 with 47 observations (20% of 233 Observations at this stage). This genotype combination was at risk level 5 not represented Risk level 2 could with 16 observations in the Merinoland and milk sheep breeds were once identified observed them in the Texel breed.

This indicates that the Amino acid expression H (histidine) at codon 154 is predominantly manifested in these two races, which is also in the ORF type ARQ / AHQ in risk class 3 with 53 observations a confirmation found the remaining two ORF types with H at position 154 (AHQ / ARH, AHQ / VRQ were in the present Data not represented. showy was the rare occurrence of genotype 144/144 in S24 with only 4 from 70 observations on ORF types with H at position 154. The ORF type ARR / ARQ risk level 3 with 165 observations found in all After ARQ / ARQ, breeds were the second most represented with 29% of 573 total observations (all observations with information on all 3 microsatellite loci and the ORF genotype). About this ORF type could still use a high proportion of the genotype combination of the microsatellites 144/144, 152/152, 179/179 with 62 observations (37.6%) become. Within this type, Locus Sil also appeared for the first time alleles 150, 154 and locus S15 allele 182 each in very low frequency. The ORF type ARR / ARH, risk level 3 with 10 Observations on codon 171 show the rare expression H (histidine). ORF types with this amino acid were in the present data with one exception in the breed Merino found only in the Texel breed (see also ORF types ARH / ARH and ARQ / ARH in risk level 4 and ARH / VRQ in risk level 5). All in all 24 animals showed this expression with a clear assignment to certain genotypes of the microsatellite loci could not be determined. The ORF type ARQ / AHQ level 3 (see ARR / AHQ and AHQ / AHQ also only represented in the Merinoland breeds and milk sheep was no further apart from the above-mentioned peculiarity striking. The ORF type ARQ / ARQ (wild type) risk level 4 with 209 observations (36.5% of all data) represented in all races, showed in terms of S24 a strong cluster of genotype 147/147 with 34% of the observations within this Type. This genotype was only 4% in risk levels 1 to 3 represented. At locus S11 a significantly higher frequency of the allele was 150 with 7% within this ORF type compared to 2% within the risk levels 1 to 3 detectable. The Locus S15 showed a significant increase in the Frequency of the allele 182 with 12.7% of the observations compared to that low occurrence of 2.3% within risk levels 1 to 3.

Alleles 150 to locus S11 and 182 to locus S15 occurred in the following risk levels with V (valine) at position 136 with even higher ones Shares on (28% share for Allel 150 and 26% share for Allele 182). However, the Locus S24 did not show any in the highest risk level conspicuity in terms of the frequencies of the microsatellite genotypes.

The present invention is accomplished by the following examples explained in more detail:

Example 1:

Materials and methods

Analysis of microsatellite motifs: Genomic DNA sequences of the genes encoding the PrP protein in GenBank (accession numbers: U67922 for sheep and AJ298878 for cattle) available and were made on microsatellite motifs using the software etandem of the EMBOSS program package (HGMP-RC, London) searched. the Motive size window was 2 to 25 nucleotides and only such microsatellite motifs with at least 3 repeats (repeats) and 90% homology evaluated. Additional microsatellite loci were created using the Sputnik program (Abajian 1994), the RepeatMasker program (Smit and Green, accessible at http://fdp.genome.washington.edu/cgibin/RepeatMasker) and the one at Lee et al. (1998) information found was a microsatellite motif Only identified in one species was the DNA sequence of the other Species through a comparison compared and the homologous area checked for the same microsatellite motif. All Sequence comparisons were carried out with the GeneDoc program, which can be found at http: // www psc.ecu / biomed / genedoc accessible is.

Animals and Samples: Blood samples were taken of 11 sheep breeds (3 animals per breed: Awassi, Changthangi, Dorper, Hu, Merinoland, Milchschaf, Gotland, Schwarzkopf, Shkodrane, Texel, White Karaman) and 8 cattle breeds (4 animals per breed: Busa, Holstein-Schwarzbunte, Jersey, South Anatolian Red cattle, Simmental, Nanjing beef, Nguni, Yerli Kara) collected.

Production of genomic DNA: DNA was stabilized from EDTA blood by chlorophore phenol Extraction or using Isolierrugskits (NucleonBAcc2, Pharmacia, Freiburg, or Nucleo Spin Blood Quick Pure, Macherey-Nagel, Düren) isolated. The quality of the DNA was checked by electrophoresis in 1% agarose gels and by photometric test at 260/280 nm. The DNA material obtained was stored in sterile TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) at -80 ° C.

Primer: The primer sequences were using the Primer 3 software (Rozen and Skletsky 1998) constructed and synthesized by Roth (Karlsruhe), whereby per locus a fluorescent primer was provided.

PCR: E3 became a standard protocol {initial denaturation for 5 min at 94 ° C followed by 30 cycles each with 1 min at 55 ° C, 1 min at 72 ° C and 1 min at 94 ° C, and a final extension for 15 min at 72 ° C.), the reaction being carried out in a volume of 20 μl with 0.15 mM primer, 200 mM of each dNTP, 100 ng genomic DNA, 1.5 mM MgCl ₂ and 0.5 units of Taq polymerase (obtained from Rotte, Karlsruhe). The efficiency and specificity of the PCR was optimized by changing the MgCl ₂ concentration (from 1.5 to 4.5 mM) together with the annealing temperature (48-65 ° C) in a gradient thermal cycler. The primers and further PCR conditions that were used for the fragment analysis according to embodiment 1 are given in Table 3.

Fragment length analysis: using of an automatic laser fluorescence DNA sequencer (A.L.F. Pharmacia) with 5% Hydrolink gels were used to combine the PCR products (0.5 μl each) with internal (99-198 nucleotides) and external (80-353 nucleotides) Standard fragments (produced by PCR using λ-DNA as a template) were analyzed. Before loading the gel, the fragments were denatured (2 min at 90 ° C and then ice cooling). Electrophoresis was performed in 0.6% TBE buffer at 1500 volts, 45 mA and 50 ° C performed. The Data analysis was carried out using the AlleleLinks software (Pharmacia, Freiburg).

Cloning of PCR products: Zur Sequencing of separate alleles were 0.067 pmol purified PCR product treated with the end conversion mix (Novagen, Madison, Wisconsin, USA) and in the EcoTV digested pT7 blue vector with blunt ends ligated. Heat shock transformation from NovaBlue E. coli host cells according to the instructions of manufacturing. Positive clones were identified by blue and white screening and in terms of the correct insert sizes Vector PCR (upstream Primers. 5'-TAATACGACTCACTATAGGG3 '; downstream primer. 5'-TTGTAAAACGACGGCCAGTG3 ') and checked electrophoresis on a 1.5% agarose gel.

Insert preparation and DNA sequencing: Template DNA was drawn from the white clones by vector PCR generated and cleaned with the Cycle Pure KIT (Peglab, Erlangen). Both strands of DNA were sequenced by MWG-Biotech (Ebersberg). The sequences obtained were created using the Chromas program (http: // www: technelysium.com.au) and GeneDoc (http: // www: psc.edu/biomed/genedoc). The Results were verified using sequencing using the Thermo Sequenase Primer Cycle Kit (Pharmacia, Freiburg) and the A.L.F. DNA sequencer analyzed and with the A.L.F. Win software package (Pharmacia, Freiburg) evaluated.

According to the present exemplary embodiment 1, 24 microsatellite loci in the bovine and 23 in the ovine PrP gene could be identified from the results in the 5 to 7 and shown in Tables 3 to 6, the following conclusions can be drawn:

Number of microsatellite loci in genomic DNA: An average of 1 microsatellite locus per 0.9 kBp DNA within the coding gene areas and the flanking Preserving regions This frequency was also observed for other coding genes and is much bigger as documented in the state of the art (vgL e.g. Schlötterer 2000). This surprising one Difference can result from the fact that in the prior art the information about Microsatellite positions in DNA mostly through screening of libraries genomic DNA elements with microsatellite motifs can be obtained and therefore the number of repeat motifs in the genome was underestimated.

Function of microsatellites in coding Genes: Repeated motifs within genes have been known for a long time. Microsatellite loci within exons indicate repetitive Codons. Regulatory DNA sequences can even be a pattern of microsatellites with even higher ones Have density, since response elements, for example, often repeat nucleotides lock in and variants in gene regulation become functionally significant (Wales et al 1990, Tripathi and Brahmachari 1991, 1995). With some Microsatellites have been involved in human genetic levels reported (O'Hara 2000, Nadir et al. 1996, Murata 2001) and occasionally the observed symptoms worsened with an increase in Number of repetitions. Christopher et al. (2000) describe a case of "repeat expansion" in one transcribed non-coding sequence that shows dysfunction which causes gene expression. It can therefore be assumed that the PrP gene found microsatellite motifs significant for gene expression and show the loci variants that are triggered by the PrP accumulation in infected cells.

Evolutionary stability of microsatellite motifs: The high intragenic variability caused by microsatellite loci can stimulate the evolution of eukaryotic genes. It was found that microsatellites in the PrP gene of humans and mice are very different (Li et al. 1996). Überraschenderwei However, in the present invention, similar positions of the microsatellite loci were found in the closely related species cattle and sheep, which positions were stable despite allelic variability within the species. This variability results in genes that are polymorphic with respect to more than one locus ("heteroalleles", whereby new alleles can be generated by intragenic recombination. In these cases, variants of individual loci that have proven successful during evolution can be recombined , which then results in new functional, possibly superior alleles.

Relationship between structure and Polymorphism of microsatellite loci: According to the invention, it was found that that microsatellites with a high number of repetitions a greater polymorphism than loci with fewer repetitions. For example were only 4 out of 25 microsatellite loci with 4 or fewer repeats polymorphic, compared to 10 out of 18 microsatellite loci with 5 and more repetitions this increase underlines the polymorphism with the number of repetitions Results described in the prior art (see, for example, Schlötterer 2000).

Allele distribution. There were microsatellite sequences examined in genetically different breeds of cattle and sheep, by as many alleles as possible to identify. In 32 cattle (from 8 different breeds) were 8 polymorphic loci (from 24 examined loci) identified. The Number of polymorphic microsatellite loci (6 out of 23) found in sheep (33 individuals from 11 races) was found to be similar to that Number in cattle, but more alleles were found in sheep, which evenly between were distributed among the races These results correspond to ORF data, where only one variant in the octapeptide motif and a silent one SNP was found in the bovine PrP-ORF, while in the ovine PrP-ORF 11 amino acid substitutions are known. The variability the ORF polymorphism is limited even in sheep because the amino acid substitutions of the relevant codons (136, 154 and 171) only in 5 different Haplotypes Occur Those depicted in the present invention In comparison, microsatellite alleles represent much more haplotypea ready and are therefore ideal for typing individuals Individuals regarding of the PrP gene.

Use of microsatellite markers in connection with ORF variants: In cattle only a few are polymorphic Jobs in the PrP gene for its typing available (Bunter 2000). As there are no informative markers in the PrP gene in the state of the Technology are known, no detailed investigation has so far been possible the relationship between BSE diposition and gene variability The new microsatellite loci are therefore becoming more genetic Examination of BSE can be used, in particular they are used for pre-diagnosis in terms of usable for this disease. In the sheep, 3 of the polymorphs Microsatellite loci used together with the ORF variants to study more than 600 animals of different breeds, what follows embodiment 3 is described.

In summary, the present embodiment 1 microsatellite loci within genes using GenBank sequences of the bovine and ovine PrP gene was analyzed. This approach is used to screen a large number of polymorphic DNA markers per gene This procedure was carried out using the PrP gene, can however, can be easily transmitted to other eukaryotic genes. When considering from microsatellites with at least 3 repetitions, 24 loci identified in the bovine and 23 in the ovine PrP gene. About 30% of the Loci were polymorphic within the species. Out of 32 cattle out of 8 genetically different races occurred 4 polymorphic microsatellites in 2.1 microsatellite with 3.1 microsatellite with 4.1 microsatellite with 6 alleles and 1 microsatellite with 10 alleles. The data from 33 sheep from 11 genetically different breeds showed a locus with 2 alleles, 2 loci with 3 alleles and 3 loci with 4 and more alleles It has been found that microsatellite loci have a higher degree of polymorphism with at least 5 repetitions, than those with 4 or fewer repetitions in cattle 9 of the 31 alleles were found only in one breed, in sheep they were 2 out of 22 alleles. frequencies of the predominant allele of <0.95 were at 6 microsatellite loci in cattle and at 6 microsatellite loci observed on the sheep. The fragment length analysis resulted in 35 of 41 comparisons with an automatic DNA sequencer by 1 to 3 nucleotides shorter Length as with sequencing. The deviations of the measured molecular Size can with secondary structures explained which are bathed by microsatellite motifs which the electrophoretic mobility of the fragment affect alleles DNA fragments differed not only in the microsatellite loci but also in their flanking ones Areas. On average, a microsatellite locus occurred in 8 genes Cattle and sheep appear approximately every 0.9 kbp, the density in PrP exons the microsatellite loci higher is. Microsatellite loci within genes can be functional in expression be and can be significant hence with the PrP accumulation in TSE-infected cells. The intragenic variability caused by Microsatellite Loci is evoked, the evolution of Eurocariotic Stimulate genes The intragenic polymorphs according to the invention Microsatellite loci particularly allow use in prediagnostics.

Example 2:

The methods set out in Example 1 were also based on the human PrP gene looking. 12 microsatellite loci were identified, 5 of which had more than 1 allele in 18 healthy people (Table 71). Polymorphic microsatellite loci can therefore also be used in humans to typify the PrP gene to identify alleles that predispose to a TSE The carriers of certain alleles can then, for example, adjust their lifestyle, in particular eating habits, to the predisposition.

Example 3:

Three of the microsatellite loci found in exemplary embodiment 1 in the region of the gene coding for prion protein (PrP gene) were tested for polymorphism in different breeds of sheep. For the investigation of the sample material, the in 8th shown method performed. In addition, the relationships between the genotypes of these loci and the ORF genotypes determined in preliminary examinations were analyzed. This is the expression of three variable positions in the Opean Reading Frame (ORF) of the PrP gene, which in many studies have shown a close relationship to the susceptibility to scrapie. From these ORF genotypes, Dawson in 1998 derived a key to the disease risk.

The data collected included 8 breeds of sheep with 623 animals from 47 flocks, collected for blood samples had been. In 573 sheep, all 3 microsatellite loci be clearly typed.

materials and methods

Animals, samples and preliminary examinations: In the trial, 623 sheep from eight different breeds and two crossing groups included (Tab. 8). Of all the sheep were Blood samples were obtained and isolated from this gDNA. From the gDNA were for the ORF of the PrP gene, the genotypes were determined (Table 8). To the result of ORF genotyping were the sheep in risk classes (Tab. 2) with regard to the likelihood of illness for scrapie assigned.

Laboratory material: The chemicals or reagents listed below or the materials listed below were used for the experiments:
Agarose, Sea Kem ^® LF, FMC organic product, Rockland, Maryland, USA
Alconox, Aldrich, Deisenhofen
Ammonium persulfate (APS), Pharmacia, Freiburg
Ampicillin, Serva, Heidelberg
Bacto-agar, Difco, Detroit, USA
Bindesilan, Pharmacia, Freiburg
Boric acid, Merck, Darmstadt
Cellulose nitrate filter SM 113 (pore size 0.45 μm), Sartorius, Göttingen
Deoxynucleotide triphosphate (dNTP mix, PEQLAB, Erlangen
Dextran blue, Pharmacia, Freiburg
DNA length standard, Gene Ruler ^TM 100 bp DNA Ladder Plus, MBI Fermentas, St. Leon-Rotte
Acetic acid, Merck, Darmstadt
Ethanol, Merck, Darmstadt
Ethidium bromide, Serva, Heidelberg
Ethylenediaminetetraacetic acid (EDTA), Merck, Darmstadt
Ficoll 400TM, Pharmacia, Freiburg
Formamid, Sigma, Deisenhofen
H ₂ O (sterile) ROTI ^® SOLV HPLC Roth, Karlsruhe
Urea, Gibco, Eggenstein
Hydrolink Long Ranger Gel Solution, Serva, Heidelberg
IPTG, Roth, Karlsruhe
Isopropanol, Roth, Karlsruhe
Potassium chloride, Merck, Darmstadt
Kimwipes, Kimberly-Clark, Koblenz
N, N, N ', N', - tetramethylethylenediamed (TEMED), Serva, Heidelberg
Perfectly Blunt Cloning Kit for pT7Blue, Novagen, Schwalbach
Pipette tips, Rotte, Karlsruhe
QIAquick Gel Extraction Kit and PCR Purification Kit, QIAGEN, Hilden
Reaction tubes (0.2 ml), PEQLAB, Erlangen
(0.5 ml, 1.5 ml, 2 ml), Eppendorf, Hamburg
Sodium dodecyl sulfate (SDS), Sigma, Deisenhofen
Taq DNA polymerase with 10-fold reaction buffer and MgCl ₂ , Eppendorf, Hamburg
Terazaki plates (MikroSample plate 60 wells), Pharmacia, Freiburg
Tetracycline, Serva, Heidelberg
Tris (hydroxymethyl) aminomethane (Tris), Merck, Darmstadt
Tryptone, Difco, Detroit
Yeast extract, Difco, Detroit
X-Gal, Roth, Karlsruhe

Aqua bidest was used to prepare the following solutions and buffers. (produced by ultrafiltration, <16 MΩ⋅cm) used. Unless otherwise stated, storage was at +4 ° C.
APS stock solution: 10% (w / v) dissolved in H ₂ O; -20 ° C
Binding silane stock solution: 40 ml ethanol; 98% (v / v); 150 ul bindesilane; RT
Binding silane solution: 800 μl binding silane stock solution, 200 μl acetic acid; 10% (v / v); RT
Hydrolink gel solution (5%): 6 ml 50% hydrolink 25.2 g urea; 3.6 ml 10X TBE; ad 60 ml H ₂ O; 200 ul APS (10%); 50 ul TEMED
Loading buffer: 50 ml formamide; 5 g mixed ion exchanger, 6 mg / ml dextran blue; 1 ml EDTA (1M); pH 8.3; -20 ° C
10 × TBE buffer: 1 M Tris-HCl 830 mM boric acid; 10 mM EDTA; pH 8.2; RT
Stop buffer: 250 mM EDTA; 10% (w / v) Ficoll 400TM; 1% (w / v) SDS; 0.05% (w / v) bromophenol blue
Ethidium bromide solution: 500 μg ethidium bromide / ml H ₂ 0 LB medium 10 g tryptone; 5 g yeast extract 5 g NaCl; 14 g agar; ad 1 l H ₂ O; autoclaved
Ampicilin: 50 mg / ml H ₂ O; -20 ° C
IPTG: 0.2 M IPTG; -20 ° C
Tetracycline: 5 mg / ml ethanol; -20 ° C
X-gal: 10% X-gal in dimethylformamide; -20 ° C

The following were used as devices or devices:
DNA sequencer: ALF (Automated Laser Fluorescence) DNA sequencer with ALTWin and AlleleLinks software, Pharmacia, Freiburg
Electrophoresis chambers: DNA-Sub-CellTM (15 × 15 cm), BioRad, Munich
Pipettes: Eppendorf "Research", Hamburg Gilson, "Pipetman", Villien-le-Bel, France
Ultrapure water system: Barnstead E-Pure, Barnstead, Dubuque, USA
Thermal cycler: PTG-200 with gradient function, MJ Research, Watertown, USA
Centrifuges: J6 B centrifuge, Beckman, Munich;
Benchtop centrifuge Biofuge A (Rotor 1386), Heraeus Sepatech, Osterode

Amplification of the microsatellites by PCR: In our own investigations, PCR approaches (Tab. 9), a PCR program (Tab. 10) and primers (Tab. 11) used the in preparatory work for the loci had been established.

Agarose gel electrophoresis of PCR products: To prepare the agarose gels, 2 g of agarose and 100 ml of 1 × TAE buffer were used in the microwave until loosened the agarose is heated with 20 μl Ethidium bromide was added and poured into the gel chamber. The slotformer was used immediately afterwards. Approximately 30 minutes later it was removed and that Gel was placed in the electrophoresis chamber using 1 × TAE buffer filled was. 5 μl PCR products were with 2 ul Stop buffer mixed and pipetted into the pockets. The separation process continued approx. 40 min. The gel was then viewed under UV light. The The result was read by comparison with the maker.

Fragment length analysis with the ALF DNA sequencer: The PCR products were separated in 5% hydrolink gels using the ALF sequencer. The control of the electrophoresis runs was carried out via the program "ALFWin Instrument Control", the subsequent evaluation with the software "AlleleLinks". The automatic sequencer consists of an electrophoresis and a detection unit. This contained a laser and 40 photodiodes, which are arranged above the individual gel tracks. Since the forward primers of the PCR and thus also the amplificates were fluorescence-labeled, they were excited when they passed the level of the laser. This led to an electrical signal in the photodiodes, which was digitized and stored in the connected computer. The fragment lengths of the individual alleles were calculated on the basis of the internal (99/198 bp) or external (additional 80/160 bp) length standards, with the AlleleLinks program converting the time measured values into bp and allowing the fluorescence signals to be displayed as a pherogram ( 9 - 11 ).

The Hydrolink gels were produced in accordance with the instructions of ALF: Manufacturer Pharmacia, Freiburg. After cleaning the glass plates with the fluorescence-free washing-up liquid Alconox, H ₂ O and 98% ethanol and subsequent air drying, the upper area was coated with binding silane in order to fix the gel pockets. The plates were then assembled with spacers and light couplers to form a gel chamber. For the gel solution, 60 ml of filtered (membrane with 0.45 μm pores) and degassed (6 min) Long Ranger Hydrolink gel solution were mixed with 200 μl APS and 50 μl TEMED. The well-mixed solution was poured between the glass plates free of air bubbles using a 60 ml syringe. A comb that was pushed into the gel chamber, was used to shape the gel pockets. After approx. 90 min the gel was polymerized at RT and could be installed in the automatic sequencers. After the gel reached 50 ° C, the comb was carefully removed. After the gel had been installed, 0.6 × TBE buffer was added to the electrode container and the gel was loaded with sample material. The gels were used three times and the gel pockets were rinsed out with TBE buffer in between. The first time gave more precise results than the following two runs

For sample preparation and application were in a Terazaki plate depending on the PCR yield 0.1-4 ul DNA with 1 ul of a mixture of length standards and stop buffer mixed (star was used 1 ul DNA). Samples were then for 2 min at 80 ° C denatured and then put on ice. After rinsing The prepared samples were pipetted into the gel pockets with TBE buffer.

The running conditions for electrophoresis were entered using the ALFWin Instrument Control software and are listed in Tab. 12.

• – Herstellung der PCR-Produkte (Locus S24) der ausgewählten Tiere (ca. 10 ng in 100 μl Lösung).
• – Reinigung der PCR-Produkte mit PCR-Purification-Kit um ca. 50 μl PCR-Produkt-Lösung zu erhalten.
• – Prüfung der DNA-Produkte auf Agarosegel hinsichtlich ihrer Qualität (saubere Banden ohne Nebenprodukte) und Quantität.
• – End-Convenion-Reaktion: Inkubation von 2 μl kondeasiertem PCR-Produkt als Template in 2 μl Wasser und 5 μl End-Conversion Mix. 30 min bei RT ruhen lassen. Inaktivierung der Mischung ca. 5 min bei 75 °C, Abkühlung 2 min auf Eis, abschließende Zentrifugation.
• – Ligation in den Vektor: 1 μl pT7Blue Blunt Vektor und 1 μl T4 DNA-Ligase wurden eingesetzt, vorsichtig gemischt und danach 1-2 h inkubiert.
• – Transformation in E. coli: 2 μl Mischung wurden in das aufgetaute "Nova Blue Single cells"-Aliquot pipettiert, danach vorsichtig gemischt und als Suspension 5 min auf Eis gelagert. Danach wurde die Suspension 35 s auf Wärmeblöcken mit einer Temperatur von 42 °C erwärmt und dann für 2 min auf Eis gelegt. 300 μl SOC-Medium wurden dazupipettiert und auf Agarplatten ausgestrichen. Die Platten wurden 24 h bei 38 °C im Wärmeschrank inkubiert.
• – Selektion der rekombinanten Klone mittels Blue-White-Screening: Die Ligationsstelle des Vektors befand sich im LacZ-Gen, das in Gegenwart von IPTG eine Blaufärbung der Kolonie bewirkte. Beim Einbau rekombinanter DNA wurde die Gensequenz unterbrochen, und die betroffenen Kolonien blieben weiß. Diese weißen Kolonien wurden mit Pipettenspitzen gepickt und in 50 μl Wasser suspendiert. Die Pipettenspitzen wurden auf eine beschriftete Platte getupft, um jeweils eine Reservekolonie herzustellen.

Cloning: In the fragment length analysis, unexpected fragments were found at locus 524. To analyze whether the DNA section of the expected microsatellite is also present in the PCR product, individual sections of the DNA molecules were sequenced. For this purpose, the "Perfectly Blunt Cloning Kit" was previously cloned in the following experimental steps:

• - Production of the PCR products (Locus S24) of the selected animals (approx. 10 ng in 100 μl solution).
• - Cleaning of the PCR products with the PCR purification kit to obtain approx. 50 μl PCR product solution.
• - Examination of the DNA products on agarose gel with regard to their quality (clean bands without by-products) and quantity.
• - End-Convenion-Reaction: Incubation of 2 μl conditioned PCR product as template in 2 μl water and 5 μl end-conversion mix. Let it rest at RT for 30 min. Inactivate the mixture for approx. 5 min at 75 ° C, cool for 2 min on ice, then centrifugation.
• - Ligation into the vector: 1 μl pT7Blue Blunt vector and 1 μl T4 DNA ligase were used, mixed carefully and then incubated for 1-2 h.
• - Transformation in E. coli: 2 μl mixture were pipetted into the thawed "Nova Blue Single cells" aliquot, then mixed carefully and stored on ice for 5 min as a suspension. The suspension was then heated for 35 s on heating blocks at a temperature of 42 ° C. and then placed on ice for 2 min. 300 μl of SOC medium were pipetted in and spread on agar plates. The plates were incubated for 24 hours at 38 ° C in an oven.
• - Selection of the recombinant clones by means of blue-white screening: the ligation site of the vector was in the LacZ gene, which in the presence of IPTG caused the colony to turn blue. When recombinant DNA was inserted, the gene sequence was interrupted and the affected colonies remained white. These white colonies were picked with pipette tips and suspended in 50 ul water. The pipette tips were spotted on a labeled plate to create a reserve colony.

Sequencing of DNA inserts recombinant vector molecules: The bacterial cell suspensions were heated to 99 ° C. for 15 min. to kill the bacteria then centrifuged and vortexed. Then a PCR was carried out at the the DNA in the reaction product as a template and the primers T7 and U19 were used (Tab. 13 to 15).

The PCR products were made using of agarose gel electrophoresis. The clones whose PCR products the expected length with the fluorescence-labeled primer pair for the locus S24 (Tab. 11) amplified and the products on the A.L.F. shown. A clone whose fragment length matched the length of 152 bp measured during genotyping, was for selected the sequencing.

A 100 ul PCR approach was performed with the primers T7 and U19 made a sufficient amount of template for the sequencing reaction to get. The length and the concentration of the PCR products was checked again on one Agarose gel tested. This was followed by the purification of the PCR products with the "PCR Purification Kit ". The samples were then sequenced.

Statistical evaluation: For frequency analysis the program package BIOSYS-1, the microsatellites and ORF data, Version 17 (Swofford and Selander 1989) used.

mit:
p_i: Frequenz des i-ten Allels,
A_iA_i, A_iA_j: Häufigkeit des Genotyps mit den Allelen i bzw. j,
i, j: Indizes der Allele,
k: Anzahl der Allele eines Locus,
N: Anzahl der untersuchten Individuen.For the co-dominant inheritance, the allele frequencies for the microsatellite loci and PrP gene loci were calculated using the following formula:

With:
p _i : frequency of the i-th allele,
A _i A _i , A _i A _j : frequency of the genotype with alleles i and j,
i, j: indices of the alleles,
k: number of alleles of a locus,
N: Number of individuals examined.

mit:
h_l: Erwarteter Heterozygotiegrad des Locus l,
p_i: Frequenz des i-ten Allels,
N: Zahl der untersuchten Individuen.The expected degrees of heterozygosity of a population for a locus with k alleles were calculated using the undistorted estimate (unbiased estimate, NEI 1978) according to the following formula:

With:
h _l : expected degree of heterozygosity of locus l,
p _i : frequency of the i-th allele,
N: number of individuals examined.

• i Homozygote Genotypen mit dem häufigsten Allel,
• ii alle heterozygoten Genotypen mit dem häufigsten Allel,
• iii alle homo- und heterozygoten Genotypen ohne das häufigste Allel.

Deviations in genotype frequencies from the Hardy-Weinberg equilibrium were checked using the pooled χ ² test. Due to the low number of observations for individual genotypes (N <5) with the problem of overestimating the significance with low genotype expected values in the χ ² test, three genotype classes (i, ii, iii) are formed in the pool method:

• i homozygous genotypes with the most common allele,
• ii all heterozygous genotypes with the most common allele,
• iii all homo- and heterozygous genotypes without the most common allele.

Multifactorial analysis of variance model taking into account the 3 microsatellite loci examined (SAS statistical package, V 6.12, GLM procedure)
Y ijkl = μ + S11 i + S24 j + S15 k + e ijkl (Model 1)
With:
Y _ijkl : characteristic value (risk class for scrapie disease) of the animal ijkl,
μ: population average,
S11 _i : fixed effect of the i-th S11 genotype,
S24 _j : fixed effect of the jth S24 genotype,
S15 _k : fixed effect of the kth S15 genotype,
e _ijkl : residual error.
(The fragment lengths given for the alleles are given below without the length unit Bp).

In embodiment 3, three the in the embodiment 1 obtained informative polymorphic microsatellite, namely S11, S15 and S24, analyzed in more detail in the breeds of Baden-Württemberg and their association with the three for scrapie susceptibility bedeutsämen ORF variants examined.

To be an inexpensive for large numbers of animals To be able to develop processes the PrP gene region was screened for polymorphic microsatellite loci (see 1st performance example). A selection of 3 from 6 suitable microsatellites was good amplifying loci hit, with a high probability a codominant inheritance was to be assumed. It became the degree of polymophy this loci within sheep breeds of Baden-Württemberg examined to based on an exam the relationships to the ORF genotypes determined from preliminary examinations and the derived crack classes with the help of the found polymorphic microsatellite loci statements about the scrapie susceptibility to be able to meet.

When analyzing the genotypes occurred no by-products, no slippage effects and no preferred Amplifications on. With two exceptions, all fragment lengths were clear each assigned to a locus-specific allele.

The first exception was the fragment length 146 at Locus S24, which occurred in a total of 6 cases with the breeds Schwarzkopf, Gotland and Suffolk. The uniqueness of the different fragment lengths 146 and 147 was ensured on the one hand by repeating the PCR application on different gels, and also by arranging animals with alleles 146 and 147 in successive traces of a gel. A fragment length difference around the precise value 1 could not be attributed to track effects if the internal marker alignment was performed at the same time. Whether fragment 146 is a very rare additional allele can only be demonstrated by sequencing. B. by deleting one Base outside the actual microsatellite area from the allele with the fragment length 147.

The second exception was fragment length 152, which was observed in 25 merino sheep. This allele never appeared homozygous but only in combination with allele 144, allele 147 or these two alleles together. In the 15 cases in which 3 peaks were observed at the same time, these always had comparable amplitudes. By cloning and subsequent sequencing of the allele 152, it was shown that this is identical to the allele 147 with regard to the microsatellite sequence ( 12 ). However, it differs in that it has a 5 bp insert on the 5 'side of the microsatellite. Further experiments were to be carried out to investigate the causes which led to the appearance of three apparently "allelic" fragments per animal.

The information content of the 3 microsatellite loci showed for 4 of the 8 breeds considered (Gotland, Suffolk, dairy sheep and Texel) mean degrees of heterozygosity> 0.35 (Tab. 19). For the breeds Dorper, Merinoland, Ile de France and Schwarzkopf were the values between 0.16 and 0.23. The information content was for the ORF loci consistently lower and showed for the mean degrees of heterozygosity between 0.0 for Gotland and 0.29 for the Texel breed. These are relatively small Information content corresponded to low observation numbers of animals with a high risk of disease. In other words, having genotypes low risk have prevailed in the populations without that those at high risk have completely disappeared. This may be due to that unfavorable PrP genotypes also cheap Cause functions in the development of an animal, e.g. B. Fertility or resistance to other pathogens.

The examination of the genotype frequencies for equilibrium situation revealed a significant deviation from the HWE with the probability of error p <0.05 only for the ORF3 locus and only for the Merinoland breed in the χ ² test. The conspicuous overrepresentation of homozygous animals can probably be explained with an average higher inbreeding coefficient, but there was insufficient family data available for testing.

The variance analysis results with model 1 (Tab. 22) with consideration the 3 microsatellite loci as fixed factors showed an almost Uniform assignment of positive or negative effects to individual genotypes in the different sheep breeds examined separately. The differences the estimated However, mean values showed significant differences. Analyzed in all 4 Breeds with animal numbers> 60 took over Locus S24 the highest Share in the declared Variance with level of significance p <0.001 (F test, Tab. 22). This locus consistently showed the homozygous type 144/144 with a lower risk of disease than the homozygous type 147/147 (Tab. 23). The distinction between these two genotypes with regard to expected disease key value varied from 0.5 in the Merino breed to 2.8 in the Suffolk breed. The regarding this locus heterozygous animals were in the risk assessment nearly in the middle between the homozygous types. The big difference in the estimated The difference in mean values was very likely to be on breed-specific ORF characteristics and on different association sizes of the genotypes of the microsatellite locus attributed to this investigated characteristic.

The locus S11 showed in the analysis of variance only a significant influence in the breeds milk sheep and Texel on the over Characterized by ORF haplotypes with pronounced risk increase for animals with the allele 150 in the Texel breed. This allele was in the breed Milk sheep for an exam not often enough represented, however, showed in this race about the key value 1 higher Risk to animals with the allele 156 opposite Animals with the allele 158 at this locus.

The Locus S15 could only be found in the Texel breed recognized a significant risk increase for animals with the allele 173 become. Corresponding to this was the information content on this Locus in the breeds Merinoland, milk sheep and Suffolk very low.

Because of the here Association with the given risk code could be a selection of sheep according to microsatellite genotypes with the characteristics 144/144 to S24, 152 / <156>, <150> S11 and 179 / <173> to S15 with a view are recommended to minimize the risk of scrapie disease (o indicates alleles to be excluded). This selection would in the present However, 47 ORF wild type observations (ARQ / ARQ with risk Include 4 This is an indication that one or two more informative microsatellites into one Elimination procedures at the genetic level should contribute if the meaningfulness a genotype combination of microsatellite loci only the basis of ORF risk coding checked becomes.

The results of performance example 3 can therefore be summarized as follows:
The ORF genotypes showed only low information levels in the sheep breeds with values between 0.0 and 0.29 for the middle degrees of heterozygosity, which resulted in a value of 0.164 for the breeds. Nevertheless, 13 of a total of 15 ORF genotypes reported in the literature were found for the total material, with approx. 65% of the observations for the ARQ / ARQ (wild type) and ARR / ARQ genotypes. The rare expression V (valine) on codon 136 with a high susceptibility to scrapie disease was only represented in this study with an allele frequency of just under 3% across the breeds.

For the 3 examined microsatellite loci could in each Breeds mean degrees of heterozygosity from 0.16 to 0.57 with a mean from 0.31 about the breeds are determined, with the Locus S24 in all breeds except Gotland the highest Showed information content.

The sorted within ORF typing Microsatellite genotypes showed a uniform risk level 1 shaping with the genotype combination 144/144, 152/152 and 179/179 for the loci S24, S11 and S15. In the higher ones Risk classes occurred in several genotype combinations. In these classes were a discrete assignment of certain microsatellite genotypes not possible for the ORF genotypes, still let clear clusters identify certain alleles in risk levels.

In the variance analysis test of Associations of the genotypes with the risk characteristic constructed from the ORF types could highly significant relationships are found. In the evaluations the Locus S24 had to the breeds with more than 60 observations the highest without exception Share in the declared Variance The estimates showed the homozygous type 144/144 in each race with a significantly lower number Disease risk out as the type 147/147, while the heterozygous animals in the estimates in between lay.

For a high degree of precision the statement on susceptibility to illness for scrapie would be after the present embodiment the inclusion of further informative microsatellite loci as well an association study with diseased and non-diseased sheep desirable.

credentials

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Abbreviations

Tab. 2: PrP-Genotypeneinteilung in Risikogruppen beim Schaf (Merkmal Risk; Dawson et al 1998)

Tab. 3A: Mikrosatellitenloci im bovinen PrP-Gen

Tab. 3B: Mikrosatellitenloci im ovinen PrP-Gen

The following abbreviations have been used in the context of the present patent application:
ALF Automated Laser Fluorescence
APS ammonium persulfate
BovB DNA repeat family from the class SINE (Short Intenpersed Elements)
Bov-tA2 / -tA3 DNA repeats from the BovA family, class SINE (Short Interspersed Elements)
Bp base pairs
BSE Bovine Spongiform Encephalopathy
CJD Creutzfeldt-Jakob disease
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
gDNA genomic DNA
HWE Hardy-Weinberg equilibrium
IPTG isopropyl-β-D-thiogalactopysnoside
LacZ gene β-galactosidase gene
mRNA messenger RNA
MS microsatellite
ORF Open reading frame
PCR polymerase chain reaction
PrP prion protein
PrP gene Prion protein coding gene
RNA ribonucleic acid
RT room temperature
Taq DNA polymerase from the bacterium Thermus aqaticus
TBE buffer made from TRIS, boric acid and EDTA
TEMED N, N, N ', N', - tetramethylethylenediamide
Tris tris (hydroxymethyl) aminomethane
TSE transmissible spongiform encephalopathy
UTR Untranslated Region Appendix Table 1: Amino acid polymorphisms in the PrP gene of sheep (after Hunter (2000), supplemented)

Tab. 2: PrP genotype classification in risk groups in sheep (characteristic Risk; Dawson et al 1998)

Table 3A: Microsatellite loci in the bovine PrP gene

Tab. 3B: Microsatellite loci in the ovine PrP gene

Tab. 8: Anzahl untersuchter Schafe und deren ORF-Genotypen

Tab. 9: PCR-Ansätze für die Loci S11, S24 und S15

Tab. 10: PCR-Programm für die Loci S11, S24 und S15

Tab. 11 Primer für die Loci S11, 524 und S15

Tab. 12: Laufbedingungen für die Elektrophorese

Tab. 13: PCR-Programm zur Sequenzierung der DNA-Inserts

Tab. 14: PCR-Ansatz zur Sequenzierung der DNA-Inserts

Tab. 15: Primer für die Sequenzierung der DNA-Inserts

Tab. 16: Fragmentlängen des Locus S24

Tab. 20: Beobachtete und erwartete Genotypfrequenzen zu den Mikrosatellitenloci der untersuchten Schafrassen mit Prüfung auf HWE

Tab. 20: Beobachtete und erwartete Genotypfrequenzen zu den Mikrosatellitenloci der untersuchten Schafrassen mit Prüfung auf HWE (Fortsetzung

Tab. 21: Beobachtete und erwartete Genotyphäufigkeiten zu den ORF-Loci der untersuchten Schafrassen mit Prüfung auf HWE

Tab. 21: Beobachtete und erwartete Genotyphäufigkeiten zu den ORF Loci der untersuchten Schafrassen mit Prüfung auf HWE (Fortsetzung)

Tab. 22: F-Werte, Signifikanzen und Bestimmtheitsmaße für Merkmal Risk zu 4 analsierten Rassen mit Beobachtungen > 60 (Auswertung gemäß Modell 1)

Tab. 23: Beziehungen zwischen Mikrsatellitenloci und dem Merkmal Risk (vgl. Tab. 2; Auswertung getrennt nach Rassen gemäß Modell 1)

Tab. 24: Beobachtungszahlen der Mikrosatellitenkombinationen innerhalb ORF-Typ

Notes on Tables 4A and 4B:
^a R: GenBank reference; bovine motifs are used for loci without GenBank information in sheep (S01 to S06); A, B, etc.: alleles found.
^b GenBank access number
^c Fragment lengths determined with the ALF sequencer
^d fragment lengths calculated based on the results of DNA sequencing
^e (R): microsatellite sequence identical to GenBank
^f -: no sequence data; kD: no difference compared to the DNA sequence in GenBank; Del .: Deletion; Ins .: insertion; SNP: single nucleotide polymorphism; *: Not all variants are specified or additional Variants are not certain
^g The breeds are indicated if an allele was found in only one breed. For a locus the sum of the alleles is less than 2n animals, if not all samples allowed a specific PCR amplification. Table 5: Polymorphism values of the investigated PrP microsatellite loci

Tab. 8: Number of sheep examined and their ORF genotypes

Tab. 9: PCR approaches for the Loci S11, S24 and S15

Tab. 10: PCR program for Loci S11, S24 and S15

Tab. 11 Primer for the Loci S11, 524 and S15

Tab. 12: Running conditions for electrophoresis

Table 13: PCR program for sequencing the DNA inserts

Tab. 14: PCR approach for sequencing the DNA inserts

Tab. 15: Primer for sequencing the DNA inserts

Tab. 16: Fragment lengths of locus S24

Tab. 20: Observed and expected genotype frequencies for the microsatellite loci of the examined sheep breeds with a check for HWE

Tab. 20: Observed and expected genotype frequencies for the microsatellite loci of the sheep breeds examined with a check for HWE (continued

Tab. 21: Observed and expected genotype frequencies for the ORF loci of the examined breeds with testing for HWE

Tab. 21: Observed and expected genotype frequencies for the ORF loci of the examined sheep breeds with a check for HWE (continued)

Tab. 22: F values, significance and certainty measures for characteristic risk of 4 analized breeds with observations> 60 (evaluation according to model 1)

Tab. 23: Relationships between microsatellite loci and the characteristic risk (see Tab. 2; evaluation separated by race according to model 1)

Tab. 24: Observation numbers of the microsatellite combinations within ORF type

Claims (37)

Method of typing a gene that contains one or more has polymorphic microsatellite loci in an individual the steps: (a) Amplify at least one DNA region of the gene to be typed by PCR with the size of the DNA region chosen Primers, the DNA region having at least one polymorphic microsatellite locus comprises and serves as a template containing a sample of the individual a DNA that has at least a portion of the gene with the polymorph (s) Includes microsatellite loci / loci, and (b) determining the length of the Amplificate / the amplificates of step (a). The method of claim 1, wherein step (6) is electrophoresis which includes amplificates. The method of claim 1 or 2, wherein the DNA region is a length of about 50 to about 500 bp. Method according to one of claims 1 to 3, wherein before step (a) determining the polymorphic microsatellite locus / loci carried out in the gene to be typed becomes. The method of claim 4, wherein determining the polymorphic (s) Microsatellite locus / loci the steps: (A) Screening the To typing gene according to microsatellite loci, (B) Amplify each of a DNA area by PCR with the size of the DNA area chosen Primers, the DNA region being that found in step (A) Contains microsatellite locus, per found microsatellite locus in a plurality of individuals the organism containing the gene to be typed, (C) Determine the length the amplificate (s) and / or the nucleotide sequence of the Microsatellite locus / loci in the amplificate (s) from step (B) for each individual and (D) determining the microsatellite locus or loci, whose amplification (s) is different between the individuals Has / have length or its nucleotide sequence (s) between the individuals is / are different. The method of claim 5, wherein step (C) is electrophoresis which includes amplificates. The method of claim 5 or 6, wherein the DNA region is a length of about 50 to about 500 bp. A method according to any one of claims 1 to 7, wherein the polymorphic microsatellite locus / loci has a sequence motif, which is repeated at least 3 times. Method according to one of claims 1 to 8, wherein the polymorphic microsatellite locus (loci has one or more sequence motifs), which are selected from the group consisting of the consensus sequences (ATA) ₇ , (GA) ₇ , (GT) ₂₈ , (AGG) ₄ , (AC) ₅ , (TTTGT) ₅ , (TA) ₇ , (TC) ₇ , (TAAA) ₁₂ , (ATTTT) ₁₂ , (TTTC) ₁₈ , (CA) ₁₈ , (CAGTT) ₄ , (AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (TTCAG) ₄ , (ACTGA) ₃ , (ACTGA) ₄ , (CA) ₁₂ , (ATC) ₄ , (T) ₂₅ , (TTG) ₅ , (T) ₂₉ , (CAA) ₃ , (TTCAG) ₃ , (AGTG) ₄ , (CAC) ₅ , (AAAACA) ₄ , (ACC) ₄ , (ATCAG) ₅ , (AC) ₃ , (CTG) ₅ , (GTT) ₅ , (T) ₁₅ , (CAA) ₄ and (AC) ₄ . Method according to one of claims 1 to 9, wherein it a gene from humans, monkeys, horses, sheep, cattle, pigs, goats, Mouse, rat, rabbit, guinea pig, dog, cat or chicken. Method according to one of claims 1 to 10, wherein the to typing gene from the group consisting of genes associated with a Disease and genes associated with milk proteins, homons or transcription factors encode, selected is. The method of claim 11, wherein the gene is from the group from PrP, CFTR, RYR1 and genes related to metabolic disorders in humans and animals, are selected. The method of claim 12, which is the human PrP gene and the sequence motif (s) of the polymorphic microsatellite locus / loci from the group consisting of the consensus sequences (ATA) ₇ , (GA) ₇ , (GT) ₂₈ , (AGG) ₄ , (AC) ₅ , (TTTGT) ₅ , (TA) ₇ , (TC) ₅ , (TAAA) ₁₂ , (ATTTT) ₁₂ , (TTTC) ₁₈ and (CA) ₁₈ , is selected. The method of claim 13, wherein the consensus sequence is (ATA) ₇ , (GA) ₇ , (TAAA) ₁₂ , (ATTTT) ₁₂ or (CA) ₁₈ . The method of claim 12, wherein the amplified DNA region (s) of the human PrP gene, nucleotides 3621 to 3641, 25415 to 25428, 39450 to 39505, 50315 to 50326, 58346 to 58385, 72392 to 72416, 78300 to 78313, 92615 to 92624, 108429 to 108476, 116907 to 116966, 125528 to 125599 and / or 147980 to 148015 according to GenBank access number AL133396 includes include. The method of claim 15, wherein the amplified DNA region (s) of the human PrP gene, nucleotides 3621 to 3641, 25415 to 25428, 108429 to 108476, 116907 to 116966 and / or 147980 to 148015 according to GenBank Access number AL133396 includes include. The method of claim 12, which is the bovine PrP gene and the sequence motif (s) of the polymorphic microsatellite locus / loci from the group consisting of the consensus sequences (CAGTT) ₄ , (TC) ₅ , (AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (TTCAG) ₄ , (ACTGA) ₃ , (ACTGA) ₄ , (CA) ₁₂ , (ATC) ₄ , (T) ₂₅ , (TTG) ₅ , (T) ₂₉ , (CAA) ₃ , (TTCAG) ₃ , (AGTG) ₄ , (CAC) ₅ , (AAAACA) ₄ , (ACC) ₄ , (ATCAG) ₅ and (AC) ₃ , is selected / are. The method of claim 17, wherein the consensus sequence is (AGC) ₅ , (TTTGT) ₄ , (CA) ₁₂ , (T) ₂₅ , (T) ₂₉ , (TTCAG) ₃ , (AAAACA) ₄ or (ATCAG) ₅ . The method of claim 12, wherein the amplified DNA region (s) of the bovine PrP gene, nucleotides 444 to 463, 4121 to 4130, 7615 to 7629, 19808 to 19817, 25288 to 25307, 30633 to 30652, 32320 to 32339, 37891 to 37910, 39943 to 39957, 44220 to 44239, 44507 to 44530, 46259 to 46278, 48409 to 48420, 50471 to 50495, 53219 to 53233, 54990 to 55018, 60452 to 60460, 62703 to 62717, 64685 to 64700, 66178 to 66192, 68762 to 68785, 68926 to 68937, 72474 to 72498 and / or 74333 to 74340 according to GenBank accession number AJ298878 comprises / comprise. The method of claim 19, wherein the amplified DNA region (s) of the bovine PrP gene, nucleotides 7615 to 7629, 25288 to 25307, 44507 to 44530, 50471 to 50495, 54990 to 55018, 62703 to 62717, 68762 to 68785 and / or 72474 to 72498 according to GenBank accession number AJ298878 comprises / comprise. The method of claim 12, wherein it is the sheep's PrP gene and the sequence motif (s) of the polymorphic microsatellite locus / loci from the group consisting of the consensus sequences (CAGTT) ₄ , (TC) ₅ , ( AGC) ₅ , (GA) ₅ , (TTTGT) ₄ , (TCAGT) ₄ , (ACTGA) ₃ , (ACTGA) ₄ , (CA) ₁₂ , (CTG) 5, (ATC) ₄ , (GTT) ₅ , ( TTG) ₅ , (T) ₁₅ , (CAA) ₄ , (CAA) ₄ , (TTCAG) ₃ , (AGTG) ₄ , (CAC) ₅ , (ACC) ₄ and (AC) ₄ . The method of claim 21, wherein the consensus sequence is (GA) ₅ , (TTTGT) ₄ , (CA) ₁₂ , (GTT) ₅ , (T) ₁₅ or (CAA) ₄ . The method of claim 12, wherein the amplified DNA region (s) of the sheep PrP gene comprises nucleotides 301 to 320, 590 to 613, 1033 to 1047, 2477 to 2496, 4651 to 4662, 6627 to 6641 , 9433 to 9447,11277 to 11291,16748 to 16756, 18100 to 18119, 19372 to 19386, 21383 to 21398, 22853 to 22867, 25422 to 25433, 25580 to 25591 and / or 30168 to 30175 according to GenBank accession number U67922 includes / comprise. The method of claim 23, wherein the amplified DNA region (s) the sheep's PrP gene, nucleotides 590 to 613, 6627 to 6641, 11277 to 11291 and / or 25422 to 25433 according to GenBank accession number U67922 comprises / comprise. Procedure for the determination of microsatellite markers for predisposition a disease associated with a gene that causes one or has several polymorphic microsatellite loci, comprising the steps: (I) Typing the gene in a plurality of individuals, of whom is known to have or not to have the disease, and / or from which it is known based on a known marker that they have a certain degree of predisposition for the Have disease by the method according to any one of claims 1 to 24  (ii) Determine genotype frequencies for each polymorphic microsatellite locus in the diseased / unaffected or a certain degree of predisposition exhibiting individuals and  (iii) Choosing the polymorphic microsatellite locus / loci as a complainer, in whom one has been diagnosed based on the genotype frequencies according to step (ii) the largest statistical Significance for the characteristics "sick" / "not sick "or for the certain degree of predisposition consists. The method of claim 25, which is a human gene, Sheep, beef, pork, goat, mouse, rat, rabbit or chicken and typing in step (i) according to the method according to one of the Expectations 10 to 24 performed becomes. The method of claim 25 or 26, wherein the gene from the group Consisting of PrP, CFTR, RYR1 and genes related to metabolic disorders in humans and animals, are associated, selected and typing in steps (i) and (ii) according to the method of a of claims 12 to 24 becomes. Prediagnostic procedure a disease associated with a gene that causes one or has several polymorphic microsatellite loci, comprising the steps: (1) Typing the gene of an individual to be examined with respect to one or more microsatellite locus / loci, which is a marker for the degree of predisposition in terms of the disease is, by the method according to any one of claims 1 to 24 and (2) Compare the genotype obtained with that Genotype-assigned degree of predisposition in terms of the disease. The method of claim 28, wherein the microsatellite cracker was determined by the method according to one of claims 25 to 27. The method of claim 28 or 29, which is a gene of humans, sheep, cattle, pigs, goats, mice, rats, rabbits or chicken and typing in step (1) according to the method one of the claims 10 to 24 performed becomes. A method according to any of claims 28 to 30, wherein the gene from the group consisting of PrP, CFTR, RYR1 and genes related to Metabolic diseases in humans and animals that are associated is selected and the typing in step (1) according to the method of a of claims 12 to 24 is carried out. The method of claim 31, wherein it is the human PrP gene and the polymorphic microsatellite locus / loci has a sequence motif selected from the group consisting of the consensus sequences (ATA) ₇ , (GA) ₇ , (TAAA) ₁₂ , (ATTTT) ₁₂ , and (CA) ₁₈ . The method of claim 31, wherein it is the bovine PrP gene and the polymorphic microsatellite locus has a sequence motif selected from the group consisting of consensus sequences (AGC) ₅ , (TTTGT ) ₄ , (CA) ₁₂ , (T) ₂₅ , (T) ₂₉ , (TTCAG) ₃ , (TTCAG) ₃ , (AAAACA) ₄ and (ATCAG) ₅ . The method of claim 31, wherein it is the sheep's PrP gene and the polymorphic microsatellite locus has a sequence motif which consists of the group consisting of the consensus sequences (GA) ₅ , (TTTGT) ₄ , (CA) ₁₂ , (GTT) ₅ , (T) ₁₅ and (CAA) ₄ . Use of microsatellite loci for typing genes. Use of microsatellite loci to identify markers in genes associated with a disease. Use of microsatellite loci for the pre-diagnosis of genes associated with are associated with a disease.