DE4138341C1 - DNA molecule for genetic pre-disposition detection to multi-endocrine neoplasia - comprises genomic cloned beta-DNA sequence and fragments in human chromosome, for hybridisation technique and selective cleavage with restriction enzymes - Google Patents

Abstract

Genomic VAC-beta-DNA and active fragments are isolated and nucleotide sequences determined by hybridisation with known fragments, selective cleavage with restriction enzymes, and identification of the prods. This material occurs at position 10q-11.2 of human chromosome 10 and is responsible for the development of multiple endocrinal tumours. USE - Used for detection of genetic pre-disposition to multiple endocrinal neoplasia type 2a (MEN-2a).

Description

The application relates to that specified in claims 1 to 5 VAC-β genomic DNA, a method for its isolation after claims 6 and 7 and their use according to claims 8 to 11.

Multiple endocrine neoplasia type 2a (MEN2a) a family cluster - autosomal dominant in inheritance - clinical picture, with simultaneous or sequential development of multiple endocrine Tumors. The most common manifestations are medullary thyroid carcinoma (MTC) and that Pheochromocytoma. The development of these tumors is similar in some other malignancies multifocal occurrence and similar inheritance, in whose Etiology so-called tumor suppressor genes play a role play (e.g. retinoblastoma). Have linkage analyzes reveal that the MEN2a locus on chromosome 10 in the Is located near the centromere. The analysis of DNA from tumors of these patients, however, gave no indication that this chromosomal region in the tumors is changed. What role does the 10q11.2 region play in the genesis of this Diseases plays is still not clear today clarified. However, it is clear that this region is affected patients unchanged to affected Offspring is passed on. DNA samples from this Chromosome areas are therefore good markers determine if this region is affected by an Was passed on to the offspring or Not. RFLP samples located in the 10q11.2 region thus represent good probes for potential gene carriers (for MEN2a).

An object of the present invention was to provide a probe in the area of MEN2a Locus hydrided on chromosome 10 (10q11.2).

Another object of the present invention was in providing a gene sample with which MEN2a Gene carriers can be recognized early.  

VAC-β / ANX8 belongs to the annexin family. To this family also includes VAC-α, lipocortin I, p67-Calelectrin and PP4-x.

Hauptmann et al. (Eur. J. Biochem. (1989) 185, 81-73) describe the cloning of the messenger RNA (mRNA) of the VACβ complementary cDNA. The mRNA can be as 2100 up to 2200 bp mRNA band detected in the Northern blot will. About VACβ genomic clones and localization the chromosomal DNA coding for VACβ in the nothing is known to the human genome.

The common structural features of the annexins are probably the basis for their similar Ca ²⁺ and phospholipid binding properties. Although this general property applies to all annexins, there is a clear individuality with regard to their affinity for Ca ²⁺ and for the different types of phospholipids.

The physiological functions of the annexins concern membrane-associated processes. The basic one Mechanism of the anticoagulant effect of the VAC has been shown to inhibit the catalytic capacity of the Phospholipids by binding the VAC to theirs Surface detected, causing the formation of the coagulation-promoting complex on its surface is prevented.

Other annexins can also inhibit blood clotting, however, VAC appears to be the most effective inhibitor.

Surprisingly, it has now been shown that the gene for VACβ on chromosome 10 near the MEN 2a locus is localized.

To isolate genomic VACβ clones, a gene bank, preferably a human blood cell bank in the vector λEMBL3, is examined for VACβ-coding sequences. For this purpose, the gene bank with a DNA coding for VACβ, e.g. B. the VACβ cDNA ( Fig. 1) or fragments of the cDNA, searched. The gene bank is plated out and hybridized with the labeled DNA probe.

The clones which can be isolated in this way can be characterized by methods known per se by restriction digestion and sequencing (Maniatis et al. (1982): "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory and Glover DM (ed.): DNA cloning , Vol. I-III, IRC Press, Oxford, Washington; (1985)). The restriction map shown in FIG. 2 is obtained by combining the results from complete and partial restriction enzyme digests. The four isolated gene clones hybridizing with VACβ cDNA cover a region of 26 kilobase pairs of human, chromosomal DNA.

The fragments of the 26 kb region which hybridize with the VACβ cDNA are cut with various restriction enzymes, cloned into a vector, for example pUC8, and the resulting plasmids (pRH301, pRH265, pRH262, pRH260, pRH254a, pRH254b and pGN49) ( FIG. prepared, the inserts isolated and using the "shot-gun method" (ultrasound treatment and cloning in phages M13mp18) according to the method described by Sanger et al. developed method sequenced (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74 (1976), 5463-5467). The DNA sequence shown in FIGS . 3A and B for the human genomic VACβ clone (Staden, R., Nucl. Acids Res. 10 (1982), 4731-4751) was then obtained by computer analysis.

For a chromosomal localization of the VACβ gene choose between two strategies. On the one hand you can use interspecific cell hybrids (human × mouse) check for the presence of these sequences. On the other hand, in situ hybridization can be used the assignment and localization of the VACβ gene to one specific chromosome area.

The first way to use cell hybrids Localization of VACβ genomic DNA used Fusion cell lines that are different human Chromosomes contained in a mouse genome. These Cell lines are selected so that a set of Cell hybrids are obtained, each being human Chromosome is present in at least two lines. To Extraction of the DNA from the different fusion lines can perform a "polymerase chain reaction" (PCR) be connected to a selective amplification that enables target DNAs. From the reaction pattern can then the corresponding chromosome on which the Target sequences are available. So that is a rough assignment of the gene to a specific one Chromosome possible (Dworzak et al. Hum. Genet., 88, 1662).

As starting cell lines for the cell hybrids z. B. a G418 resistant form of the HGPRT negative mouse Myeloma line P3X63-Ag8.653 (ATCC CRL 1580) was prepared will. For example, come as humane fusion partners the Ewing cell lines SAL-2 and STA-ET-2.1 or normal human lymphocytes in question. The corresponding cells are fused under conditions known per se and dressed. You will then not be using a  radioactive in situ hybridization (ISH) analyzed, to make the human chromosomes unique in this way to be distinguished from the mouse chromosomes and to be able to identify. These in situ hybridizations become more human with biotinylated Total DNA performed as a sample. The hybridization areas can then with known antibody reactions be made visible. In addition to the hybridization signals becomes a fluorescence R-band method used to make the human chromosomes unique to be able to identify.

The "polymerase chain reaction" (PCR) is a method for the propagation of DNA in vitro. At a double-stranded DNA as a template bind two primers, whose 3'-ends point towards each other. The DNA is at 94 ° C denatured. The primers bind when they cool to 55 ° C to the template. This reaction is opposite to that Rehybridization is kinetically preferred because the primers in Excess are used. Heat stable DNA Polymerase from Thermus aquaticus (Taq polymerase), extends the primer in Presence of deoxynucleoside triphosphates at 72 ° C. The 94 ° C-55 ° C-72 ° C cycle becomes 10- to as needed Repeated 40 times. The concentration of the DNA section delimited by the two primers theoretically exponentially too. At the end of the PCR there is µg Amounts of the DNA to be amplified.

Being a very complex DNA like genomic DNA Template used, there is a good chance that the used primers not only at the desired one  Tie position. Through non-specific binding of the PCR primers can be of a variety of different types Reaction products come. To the specificity of the To increase response, the PCR response can occur early be broken off before still on agarose gels visible amounts of DNA products arise. The Reaction solution is diluted and a second, oligonucleotide pair binding within the first pair of primers is added as a primer. In a second PCR specifically binds the second pair of primers the already amplified, desired DNA fragment, but also unspecific to the original as a template Genomic DNA used. But since the amplified DNA fragment is already in large excess, a few cycles are sufficient for the second PCR to do this generate the desired DNA fragment in an amount that can be detected on agarose gels. The Concentration of the non-specific also arising DNA fragments are still far below the detection limit.

Eight oligonucleotides specific for the VACβ gene were synthesized and purified by the OPC method. Two pairs of oligonucleotides each form a nested set of primers for PCR. The first set binds in intron 5 at the border to exon 5, the second set lies entirely in intron 5. The primers used are shown in FIG. 4.

DNA of selected hybrid lines ( Fig. 6) is isolated and subjected to a "polymerase chain reaction". The reaction products are analyzed by electrophoresis in an agarose gel ( Fig. 5). It turns out that only cell lines that contain the human chromosome 10 produce reaction products ( FIG. 5). This shows that the gene for VACβ is localized on human chromosome 10.

In order to be able to make an exact statement on which specific section of this chromosome the gene is located, the non-radioactive in situ hybridization technique using biotin-labeled DNA samples was used. With this technique it is possible to localize the VACβ gene on the chromosome section 10q11.2 (Ex. 5 ).

The ISH using peroxidase detected sample and the reflectance contrast representation in Fig. 8A and the corresponding R-banded human chromosomes 8 B are shown in Figure. The quantitative distribution of the hybridization signals on human chromosome 10 is shown in FIG. 7.

The peroxidase signals are arranged on both chromatids of chromosome 10 in the pericentromer region 10q11.2.

Thus, the genomic VAC-β according to the invention Gen provided a probe in the area of MEN2a locus hybridizes. By comparing the Hybridization pattern of the VAC-β gene with DNA samples from Relatives can be told whether the examined patient / subject is a gene carrier for MEN2a or not. This information leaves an exact  Estimating the risk of whether this patient is at one typical malignancy for this clinical picture will or not. For this, DNA samples from Subjects / patients isolated according to known methods, digested with specific restriction enzymes and with the VAC-β sample, or a sequence from the immediate neighborhood of the gene under usual Conditions hybridized. In the same way with proceed with the DNA samples of all available relatives. The RFLP analyzes can of course also be carried out using the PCR technology can be performed.

Due to restriction fragment length poly morphisms (RFLP) within or in the immediate Neighborhood of the VAC-β is a DNA sample provided that enable these studies.

Example 1: Isolation of VACβ gene clones. Cultivation of the E. coli Mg ⁺⁺ cells

E. coli is grown in LB medium (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl) containing 0.2% maltose. In the logarithmic growth phase, the absorbance at 600 nm is determined, the bacteria are centrifuged off, the supernatant is removed, and the bacteria are taken up in 10 mM MgSO₄ to a density of 2 OD _{600 nm} / ml.

Manufacture of nitrocellulose filter prints

It becomes a human blood cell gene bank in the vector λEMBL3 USA; Frischauf, A.-M., Lehrach, H. Poustka, A. and Murray, N., J. Mol. Biol. 170 (1983), 827-842) with approximately 4 × 10⁶ independent clones. Approximately 100,000 phages in 20 µl λ diluent (10 mM Tris, pH 8.0, mM MgCl₂, 0.1 mM EDTA) are mixed with 300 µl E. coli NM 539 Mg ⁺⁺ cells (2 OD _{600 nm} / ml) in 6.5 ml top agarose (7 g / l agarose, 10 g / l tryptone, 5 g / l NaCl, 5 g / l yeast extract, 10 mM MgSO₄) on LB agar (15 g / l agar, 10 g / l trypton , 5 g / l NaCl, 5 g / l yeast extract, 10 mM MgSO₄) plated in 14.5 cm petri dishes. A total of 21 plates are produced in this way.

The plates are incubated at 37 ° C for 16 hours then held at 4 ° C for 1 hour. From every plate are two prints on nitrocellulose filters produced. The filters are in at room temperature  treated the following solutions: 1 minute in 0.5 N NaOH / 1.5 M NaCl, 5 minutes in 0.5 Tris pH 8.0, 1.5 M NaCl and 30 seconds in 2 x SSPE (2 x SSPE: 0.36 M NaCl, 20 mM sodium phosphate, pH 7.4, 2 mM EDTA). The Filters are air dried and then at 80 ° C baked.

Pitch translation

The plasmid pRH 203, which contains the VACβ (base 1 to 1012 in Fig. 1, EcoRI ends, cloned into the EcoRI site of pUC 8) (Hauptmann, R. et al., Eur. J. Biochem. 185 (1989) 63-71), is linearized with HindIII. For this purpose, 1 µg pRH 203 in 30 µl medium buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 50 mM NaCl, 100 µg / ml BSA (bovine serum albumin), 1 mM DTT (dithiothreitol) with 9 units of HindIII for one hour Incubated at 37 ° C. The digestion is ended for 5 minutes by heating the solution to 70 ° C. 1 μl of 1 mM dATP, dGTP and dTTP, 10 μl of α-32 P-dCTP PB 10204, 370 MBq / mmol, are added to the solution. 10 mCi / ml), 2 µl 10 × nick translation buffer (10 × buffer: 500 mM Tris / HCl, pH 7.5, 100 mM MgSO₄, 1 mM DTT (dithiothreitol) 500 µg / ml BSA (bovine serum albumin), 5.5 µl Water and 1.5 µl DNA polymerase I (7.5 units added, and the solution incubated for 2 hours at room temperature. The reaction is stopped by adding 2 µl 0.5 M EDTA solution, and the non-built-in radioactivity by Sephadex G100 column chromatography This approach is carried out four times and the eluates from the column chromatography are combined in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) about 100 × 10⁶ cpm³²P built into the DNA.

Filter hybridization

The 42 filters are in a solution of 50 mM Tris pH 8.0, 1 M NaCl, 1 mM EDTA and 0.1% SDS three times for 1 hour Pre-washed at 65 ° C (0.5 l each).

Then they are two hours at 65 ° C in 120 ml 5 × SSPE, (0.9 M NaCl, 50 mM sodium phosphate, pH 7.4 5 mM EDTA) 5 × Denhardt's (1 g / l Ficoll, 1 g / l polyvinyl pyrrolidone, 1 g / l bovine serum albumin) 0.1% SDS pre-hybridized. The nick-translated DNA is replaced by 5 minutes at 100 ° C denatured and immediately in pipetted in the 120 ml prehybridization solution. The Hybridization took place at 65 ° C for 18 hours.

The filters are washed extensively: three times 10 minutes at room temperature in 2 × SSC (1 × SSC: 150 mM NaCl, 15 mM sodium acetate, pH 7.5), 0.1% SDS and three times at 65 ° C in 2 × SSC, 0.01% SDS. After this The filters are air dried at -70 ° C for 24 hours Kodak X-OmatS film exposed.

Plaque cleaning

Plaques showing the hybridization probe binding on both filters are pricked out with the thin end of a Pasteur pipette. The agar plug is eluted in 0.5 ml λ buffer (100 mM Tris / HCl, pH 7.5, 10 mM MgSO ₄ , 1 mM EDTA), mixed with 50 μl chloroform, at 4 ° C. for one day.

The eluates are diluted 1: 200 with SM buffer (100 mM NaCl, 10 mM MgSO ₄ , 50 mM Tris / HCl, pH 7.5, 0.01% gelatin), 10 ul of which with 100 ul E. coli NM 538 (ATCC 35638, F ^- , subF, hsdR, mcrB, λ ^- ) Mg ⁺⁺ cells and 3.5 ml top agarose plated on LB agar (9.5 cm petri dishes) and incubated for 16 hours at 37 ° C.

Two nitrocellulose filters are placed on each plate deducted, which were treated as described above.

Hybridized plaques are again cut out and processed as previously described.

After a total of three rounds of plating and hybridization the hybridizing λ clones are isolated.

Phage strain suspension

100 µ. E. coli NM 538 Mg ⁺⁺ cells are plated with 25 ul phage eluate of a homogeneous plaque and 3.5 ml top agarose on LB agar. After 16 hours of incubation at 37 ° C., the agar is covered with 5 ml of SM buffer, and the phages are eluted at 4 ° C. for 6 hours. The supernatant is filtered off with suction, mixed with 100 ml of chloroform and centrifuged for 10 minutes at 4500 rpm in a J2-21 centrifuge. The clear supernatant is carefully removed, mixed with 40 µl chloroform and stored at 4 ° C.

A total of 33 λ clones were isolated, which with the Hybridize VACβ cDNA probe.  

Example 2: Characterization of the λ clones Production of λ DNA

Approx. 2.5 × 10⁶ pfu of a λ clone are plated with 300 μl E. coli NM 538 Mg ⁺⁺ cells and 6.0 ml top agarose on LB agar and incubated for 5.5 hours at 37 ° C. Totally lysis of the E. coli bacteria is achieved; the plaques are confluent. The phages are eluted for 16 hours at 4 ° C with 10 ml λ diluent. The supernatant, about 8 ml, is removed and transferred into 15 ml Corex tubes. The suspension is centrifuged for 10 minutes at 15,000 rpm in the J2-21 centrifuge at 4 ° C in the JA20 rotor. The supernatant is decanted into polycarbonate tubes. The λ phages are pelleted by centrifugation at 50,000 rpm, 20 ° C in a 50 Ti rotor to ω²t = 3 × 10¹⁰ (about 23 minutes). After removing the supernatant, the phages are resuspended in 0.5 ml λ diluent. The suspension is transferred to 1.4 ml Eppendor tubes and non-resuspended portions are removed by brief centrifugation. RNase at 10 µg / ml and DNase at 1 µg / ml are added and incubated at 37 ° C for 30 minutes. 25 µl 0.5 M EDTA, 12.5 µl 1 M Tris, pH 8.0 and 6.5 µl 20% SDS are added to the suspension. Proteins are removed by phenol extraction and the λ DNA is precipitated from the aqueous phase after the addition of 1/20 vol. 3M sodium acetate and 1 ml of ethanol. The DNA is pelleted by centrifugation, washed once with 70% ethanol, briefly dried in a vacuum and finally dissolved in 50 µl TE buffer.

Complete restriction enzyme digestion

Each 1 µg λ DNA are according to the instructions of the manufacturer with the Restriction enzymes EcoRI, HindIII, BamHI and SalI cut, and the DNA fragments on a 0.7% Agarose gel separated in TBE buffer. By comparison the pattern revealed that some DNAs showed identical sewing patterns. Overall, could four different classes of patterns be identified (class I-IV).

Partial restriction enzyme digestion

To get a map of the restriction enzyme sites of the to create different clones must be done after Restriction enzyme cut the resulting DNA fragments be ordered. This is done by partial Cutting the λ DNAs (Rackwitz, H.R., Zehetner, G., Frischauf, A.-M. and Lehrach, H., Gene 30 (1984), 195-200).

100 ng oligonucleotide cosR (complementary to the cohesive End of the short λ arm (sequence: 5′-GGGCGGCGACCT-3 ′, made on an Applied Biosystem Model 381 DNA Synthesizer according to the manufacturer's instructions) are in 10 ul 70 mM Tris, pH 7.5, 10 mM MgCl₂, 5 mM DTT (dithiothreitol), 150 µCi γ-32 P-ATP, 10 units of T4 polynucleotide kinase Phosphorylated for 30 minutes at 37 ° C. After adding 10 µl 50 mM EDTA is not incorporated by γ-32 ATP Chromatography on a Biogel P6DG column separated. The eluate with the radioactive cosR showed 431,000 cpm per µl.  

4 µl of the λ DNA are combined in a total of 30 µl together with 1 µg herring sperm DNA as a carrier with 2 units of EcoRI (in high buffer: 50 mM Tris, pH 7.5, 10 mM MgCl ₂ , 100 mM NaCl, 100 µg / ml BSA, 1 mM DTT), 2 units HindIII or 2 units BamHI (each in medium buffer: 50 mM Tris / HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl ₂ , 100 µg / ml BSA, 1 mM DDT ) cut. After 15, 30 and 45 minutes at 37 ° C., 10 μl aliquots are removed and pipetted into 6 μl stop solution preheated to 70 ° C. (stop solution with EcoRI digestion: 250 mM EDTA, with HindIII and BamHI: 250 mM EDTA, 250 mM NaCl ). The combined aliquots are kept at 70 ° C. for a further 5 minutes and brought to room temperature. 50 µl of the cosR solution are mixed with 1 µl 5 M NaCl. 5 ml of this are mixed with 5 µl of the stopped partial digestion solution, incubated for 3 minutes at 70 ° C and 45 minutes at 45 ° C. The radioactively labeled oligonucleotide is hybridized to the cos end of the short λ arm. The fragments are separated on a 0.45% agarose gel in TBE buffer (10.8 g / l Tris, 5.5 g / l boric acid, 0.93 g / l EDTA) at 1 V / cm for 60 hours. Radioactive labeled λ DNA cut with EcoRI, HindIII or SalI are used as markers. The agarose gel is dried and exposed to Kodak X-Omat S film.

The restriction interface maps in FIG. 2 are created by combining the results from complete and partial restriction enzyme digests . The four isolated gene clones hybridizing with VACβ cDNA cover a region of 26 kb (kilobase pairs) of human chromosomal DNA.

Example 3: Sequence according to VACβ gene.

Fig. 2 also shows which areas of the genomic DNA hybridize with the VACβ probe. The λ DNAs of corresponding classes are cut twice with BamHI and HindIII, HindIII and EcoRI or only with EcoRI, the hybridizing DNA fragments are isolated and ligated and cloned with correspondingly prepared pUC 8 DNA. The plasmid DNA of the resulting clones pRH 301, pRH 265, pRH 262, pRH 260, pRH 254a, pRH 254b and pGN49 ( FIG. 2) is prepared in a larger amount. The inserts were isolated and sequenced using the "shotgun" method.

Approximately 10 µg of the DNA is ligated in 50 µl reaction solution using T4 DNA ligase, the volume is brought to 250 µl with nick translation buffer, and the DNA is then broken up using ultrasound (sonicator water bath, 5-second treatments, 1 minute incubation on ice in between) . The ends of the fragments are then repaired for two hours at 14 ° C. by adding 2.5 μl of 0.5 mM dATP, dGTP, dCTP and dTTP and 10 units of Klenow fragment of DNA polymerase I. The resulting straight-ended DNA fragments are separated in size on a 1% agarose gel. Fragments of sizes between 500 and 1000 base pairs are isolated and subcloned into the SmaI cut, dephosphorylated phage vector Ml3mp8. The single-stranded DNA of recombinant phages is isolated and sequenced according to the method developed by Sanger (Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74 (1976), 5463-5467). The individual sequences were combined to form the overall sequence using the computer programs originally developed by Staden (R. Staden, Nucl. Acids Res. 10 (1982), 4731-4751; FIGS . 3A and 3B).

Example 4: Chromosonal localization of the human VACβ gene Creation and testing of the hybrid cell lines

The hybrid cell lines were according to Dworzak et al. (Hum. Genet., 88, 1662).

All cell lines are in RPMI 1640 with 10% FCS, Glutamine and penicillin / streptomycin cultured. As starting lines for the cell hybrids becomes a G 418 resistant form of HGPRT negative Mouse myeloma line P3 × 63-Ag8.653 (ATCC CRL 1580) produced. Two become human humane partners Ewing tumor cell lines SAL-2 and STA-ET-2.1 or normal human lymphocytes are used. There will be 1-2 × 10⁷ Cells of each fusion partner mixed together, washed and in the presence of preheated PEG (0.5 ml a 41.7% PEG 4000 solution constant motion fused. As a selection medium serves RPMI 1640 supplemented with 2% HAT and 500 µg / ml G418. After two to three weeks the clones are growing on fresh medium implemented. The clones are partially frozen and on the other hand by means of non-radioactive in situ Hybridization (ISH) analyzed.  

DNA extraction from the fusion lines

The DNA salting-out technique according to Miller, S.A., Dykes, D.D. and Polesky H.F. (1988), Nucleic. Acid. Res. 16, 1215) was used.

Identification of the human chromosomes in the fusion lines

In order to clearly distinguish the human chromosomes from the To be able to distinguish mouse chromosomes and around them Being able to clearly identify them becomes one non-radioactive in situ hybridization (ISH) with biotinylated total human DNA. The Hybridization areas are marked using TRITC Antibodies made visible. First antibody: mouse anti-biotin 1:20, second antibody: rabbit anti-mouse F (ab ′) ₂-TRITC 1: 100 (both Antibodies in 3% BSA / PBS; 40 minutes incubation time at 37 ° C in a humid chamber followed by a 10 minute wash in 0.1% BSA / PBS after each Incubation step). In addition to hybridization signals a fluorescence R-banding method was used to measure the to be able to clearly identify human chromosomes.

Polymerase chain reaction (PCR)

In 50 ul reaction solution 100 ng of genomic DNA from the cell hybrids ( Fig. 5 and 6) with 50 pmol Primer 1 and 50 pmol Primer 2 in the presence of 50 mM KCl, 10 mM Tris, pH 8.3, 1.5 mM MgCl₂ , 0.01% (w / v) gelatin, each 0.2 mM dATP, dGTP, dCTP and dTTP and 1.25 units of Taq polymerase amplified. A Perkin Elmer Cetus DNA Thermal Cycler can be used with the following programming: 5 minutes 94 ° C (delay), followed by the step cycle: Step 1: 40 seconds 94 ° C, Step 2: 45 seconds, 55 ° C, Step 3 : 90 seconds 72 ° C. 40 cycles, 3 seconds extension per cycle at step 3. Finally there are two delay files: 7 minutes 72 ° C and 2 minutes 25 ° C. The procedure is completed with a soak file (4 ° C). The reaction solution is then diluted 1: 100 with water.

2 µl of these are used as templates in the second PCR. The reaction solution (50 μl) contains 50 pmol of primer 3 and 50 pmol of primer 4 ( FIG. 3), as well as the other components as described above. The temperature cycle is also the same, but only 15 step cycles are carried out.

The reaction products are on a 1% agarose + 2% Nutella agarose gel separated in TBE buffer.

As a comparison, λ DNA containing intron 5 was subjected to the same PCR procedure. The result is shown in FIG. 5. One recognizes reaction products with DNA from XXD1, XXB2 and XXM cell lines as starting material. Fig. 6 shows the evaluation of the result. These three cell lines are the only ones in which the human chromosome 10 could be detected. This shows that the gene for VACβ is localized on human chromosome 10.

Example 5: In Situ Hybridization (ISH) Incorporation of nucleotides

The DNA sample is mixed with the corresponding nucleotides and enzymes mixed and the nick translation is carried out using a kit for two hours at 15 ° C with the addition of bio-11-dUTP, dGTP, dCTP and dATP with a reaction mixture of 100 µl, carried out according to the manufacturer's instructions.

Check the installation

It will be a dot blot in 1:10 Dilution steps to quantify the marked Sample produced. The visualization takes place by means of alkaline phosphatase coupled streptavidin (Blue gene detection system). Dots with 0.02 pg or less biotinylated sample DNA should be clearly visible.

Pretreatment of the slides and chromosome preparation

Slides are made before dropping the Cell suspension in fresh methanol / acetic acid (3: 1); for 10 min. put and with a chemically inert Wipe the cloth briefly dry. Through extended treatment of the cells with hypotonic KCl solution for up to 45 minutes 37 ° C and / or more often fixing in fresh, chilled Methanol / acetic acid are absolutely free of cytoplasm Preserved metaphases.

Storage of the chromosome preparations

Warehousing takes place in the absence of air at -20 ° C or -70 ° C for a few weeks to a year.

RNase treatment

The dry preparations are with DNase-free RNase (100 µl / ml in 2 × SSC) for one  Incubated at 37 ° C for one hour. The preparations are then through an ascending alcohol series (70%, 96% and 100%) dewatered and in one Desiccator dried. The dehydrated drugs are overlaid with 20 µl hybridization mix and covered with a cover slip (24 × 60 mm). The Hybridization mix consists of the following components: 100 µl formamide (deionized with Bio-Rad AG 501-X8); 40 µl 50% dextran sulfate; 20 µl 20 × SSC, pH 7; 1 µl dissolved biotinylated DNA sample (linearized plasmid in a final concentration of 2 µg / 50 µl and 5 µl treated with ultrasound and denatured herring sperm DNA (10 mg / ml); Final volume 200 µl).

De- and renaturation

The marked sample will denatured together with the chromosome preparations: 20 µl of the hybridization mix are used per preparation applied and cleaned with an alcohol (ethanol) Cover glass (24 × 60 mm) covered. After the Has spread liquid film, the cover slip sealed with rubber cement. After this The preparations are dried in a damp chamber denatured at 79 ° C in metal boxes. After this The metal boxes are denatured into one Incubator transferred and added for 2 to 10 hours 37 ° C renatured.  

Washing steps

The rubber cement is peeled off Preparations are made in 2 × SSC at room temperature set. It is done twice for 5 minutes each in 2 × SSC at room temperature, three times for 5 minutes each in 2 × SSC 37 ° C, once for 3 minutes in PBS with 0.1% Triton X-100 and three to five times thoroughly with PBS without Triton washed.

Detection method

The preparations are mixed with 500 µl 3% BSA (bovine serum albumin) or human serum under pre-incubated in a coverslip for 3 minutes. The Preparations are made with 100 µl primary antibody (rabbit-anti-biotin, 1: 1000) in 3% BSA / PBS layered, covered with coverslip and for 30 minutes incubated at 37 ° C, then 2 × 5 minutes in 0.3% BSA / PBS washed. The preparations are with biotinylated antibody incubated at 37 ° C for 30 minutes (bio-goat-anti-rabbit; 1: 1000 in 3% BSA in PBS). Then washed 2 × 5 minutes in 0.3% BSA / PBS and 30 Minutes at 37 ° C with streptavidin peroxidase or anti-biotin antibodies (e.g. Detek I-hrp ENZO DS 820-1; Dakopattts P 397) 1: 1000 in 1 × Detek buffer or 3% BSA / PBS) incubated. Then others follow Washing steps: 3 × 5 minutes with 2 × SSC, 1 × 3 minutes PBS including 0.1% Triton X-100; 3 to 5 × briefly with PBS. Peroxidase is developed using DAB (Dianisidino-2-phenylindole).

Microscopic analysis

The preparations are not included, but directly with one Considered immersion lens. The use of the Reflection contrast method - as described by Landegent et al. (Landegent, J.E. et al. (1985) J. Histochem. Cytochem. 33, 1242-1246  described - allows the representation of very small Amounts of peroxidase precipitates. The used Microscope is for example a Leitz / Leica Fluorescence microscope (Ortholux II) with a Ploem opaque Incident lighting that contains a pole filter system and an oil RK lens 50 × to achieve the Reflection contrast. They are photographed labeled chromosomes using a Variomat Photo equipment on a technical pan Movie. A is used for chromosome identification modified CDD staining protocol used (Swiss, D. (1981) Hum. Genet. 57, 1-14).

Chromosome identification

The combined representation of peroxidase ISH signals and chromosome bands takes place according to the instructions of Ambros P. F. and Karlic, H. (1978) Hum. Genet. 77, 251-254. The coloring and Fixation with chromomycin and paraformaldehyde is used performed overnight in a humid chamber. To The triple staining is done with a Glycerin solution covered (Glycerin / Mc Ilvaine, pH 7, 1: 1, including 2.5 mM MgCl₂) with rubber cement included and "aged" for three days at 37 ° C to stabilize the fluorescence. The corresponding Mitoses are made with a 100 Pl Fluotar lens photographed.

evaluation

A total of 28 mitoses were analyzed with the VAC-β probe and a total of 185 signals were counted. In the chromosome region 10g11.2, the most signals are counted based on the band size (17.93%). In contrast, 43.48% of the signals were assigned to the chromosome bands 10p11.2-q21.1 ( FIG. 7).

Images of an ISH sample detected using peroxidase and reflection contrast representation ( FIG. 8a) and the corresponding R-banded human chromosomes can be seen in the figure ( FIG. 8b). The peroxidase signals are arranged on both chromatids of chromosome 10 in the pericentromer region 10q11.2.

Legend to the pictures

Fig. 1 cDNA and deduced amino acid sequence of VACß (Annexin VIII).

Fig. 2 upper part: restriction maps of the four different λ classes, which were butted using the VACß cDNA probe (B: BamHI, E: EcoRI, H: HindIII). Lower part:. Restriction fragments subcloned into pUC vectors for the purpose of sequencing and their names. The twelve exons are entered, which together comprise the entire area of the sequence determined from the cDNA.

Fig. 3 Sequence of the genomic VACβ gene. The sequences in Figures 3A and 3B are separated by some nucleotides, the sequence of which has not been determined.

Fig. 4 PCR primer sequences for the determination of the human chromosome that contains the VACβ gene.

Fig. 5 Upper part: Photo of the agarose gel with the result of the PCR to determine the VAC-beta gene containing human chromosome. Upper section of the gel: use of the 1st set of primers, lower section: second set of primers. Lower part: schematic representation. M :. Size marker, λ: recombinant λ DNA, which contains the VAC-beta intron 5, 1-9: template DNA from different mouse-human hybrid cell lines: 1, DU C2, 2, KK D1, 3, C1 SAL, 4, DU Cl 5, XX B2, 6, XX Al, 7, DU C5, 8, XX C5, 9, XX M.

FIG. 6 Evaluation of the result from FIG. 5. One line contains the information as to which human chromosome is contained in the corresponding cell line. An empty box means that the corresponding chromosome was not found. The numbers and letters above the columns indicate the respective chromosome. The column "N" means that parts of the chromosome have been found which cannot be clearly assigned to a specific human chromosome. A line highlighted in gray means that DNA from this cell line has delivered a PCR product. The gray column "10" means that only chromosome 10 can be responsible for the PCR pattern obtained.

Fig. 7 shows a schematic representation of the distribution of hybridization signals on chromosome 10.

Fig. 8A and B :.

In situ hybridization with a medium Peroxidase detected sample in the form of a Reflection contrast display (A) and the corresponding R-banded human Chromosomes (B). The peroxidase signals are each on both chromatids of the chromosome 10 in the pericentromer region 10q11.2 arranged.

Claims (13)

1. A DNA molecule, characterized in that it comprises the genomic shown in Fig. 3A and 3B VAC-β-DNA, in which Fig. 3A and Fig. 3B are part of this claim and fragments of the sequence shown in Figures 3A and Fig. 3B . 2. DNA molecule according to claim 1, characterized characterized it on human Chromosome 10 is localized. 3. DNA molecule according to claim 1, characterized characterized it on human Chromosome 10 is located at position 10q11.2. 4. DNA molecule according to claim 1, characterized in that it by

• a) Search for a genomic phage DNA bank
• b) hybridization with a DNA fragment or partial fragment coding for an exon region of the VACβ gene and
• c) separation of the phages thus obtained and
• d) Characterization of the DNA by restriction analysis and / or sequencing

is obtained. 5. DNA molecule according to claim 1, characterized in that it represents one of the plasmids shown in Fig. 2 pRH301, pRH265, pRH262, pRH260, pRH254a, pRH254b and pGN49, with Fig. 2 being part of this claim. 6. A method for isolating a genomic VACβ DNA molecule according to claim 1, characterized in that

• a) a genomic phage library with a nucleic acid coding for VACß by
• b) hybridization is searched and
• c) after separation of the phages thus obtained
• d) the DNA by restriction analysis and / or sequencing

is characterized. 7. The method according to claim 6, characterized in that that the genomic gene bank the whole human genome. 8. Use of a DNA fragment according to claims 1-5 as a means for hybridization to a DNA sample, characterized in that

• a) the DNA fragment is present as a single-stranded, labeled, denatured DNA and
• b) is hybridized to a DNA sample which
• c) comes from a human tissue sample,
• d) is cut with one or more restriction enzymes and
• e) is fractionated and denatured by size.

9. Use of a DNA fragment according to the Claims 1-5 and 8 as a means of detection genetic predisposition of multiple endocrine neoplasia type 2a (MEN2a).   10. Use of a DNA fragment according to the Claims 1-5 and 8 as a means of detection multiple endocrine neoplasia type 2a (MEN2a). 11. Use of a DNA fragment according to the Claims 9 and 10, characterized in that it when analyzing a restriction fragment length polymorphism (RFLP) is used.