WO1995030771A1

WO1995030771A1 - Process for analysing chromosome dna

Info

Publication number: WO1995030771A1
Application number: PCT/EP1995/001706
Authority: WO
Inventors: Andreas Weith; Christoph Lengauer
Original assignee: Boehringer Ingelheim International Gmbh
Priority date: 1994-05-10
Filing date: 1995-05-05
Publication date: 1995-11-16
Also published as: DE4416393A1

Abstract

The proposed processes for analysing chromosomes are based on non-radioactive methods, in particular fluorescence in situ hybridization, whereby a P1-clone-DNA is used as a hybridization probe. These processes can be used above all in clinical and prenatal diagnosis and in tumour-interphase cytogenetics.

Description

Method for examining chromosomal DNA

The present invention relates to a method for examining sections of the mammalian genome, in particular the human genome.

The cloning of chromosomal subregions to saturation depends on the complexity and quality of the libraries containing long clones used for hybridization screening. To date, Cosmide (Murray et al., 1983) and so-called "Yeast Artificial Chromosomes" (YACs; Burke et al., 1987) have been the preferred vector systems routinely used for cloning high molecular weight DNA. However, each of these systems has drawbacks. Since cosmid vectors do not accept DNA inserts larger than 40 kb (Whittaker et al., 1988), their use for the analysis of a large gene region is subject to restrictions. The YAC clone system allows much larger inserts (up to 1 Mbp); However, studies have shown that high frequency YAC clones (e.g. Green et al., 1991) are chimeric, i.e. contain fragments that do not belong together within a clone, and / or show deletions and internal rearrangements.

The clones available to date are also subject to restrictions in their use in diagnostic methods, in particular non-radioactive in situ hybridization methods: cosmid clone fragments are often too short, so that the label attached is sometimes insufficient to obtain evaluable signals; the YAC clones, on the other hand, often give diffuse signals due to their long length. The object of the present invention was to provide a new method based on non-radioactive in situ hybridization.

To solve the problem, a human library was first tested for its suitability for hybridization screens, about which no information is available to date. The PI cloning system was designed as an alternative to the cosmid and YAC cloning systems (Sternberg et al., 1990; Pierce and Sternberg, 1992); it uses the bacteriophage Pl as a vector, which enables the cloning of inserts up to 100 kbp in size. The human PI library was first used for hybridization screening in the context of the present invention, 80 microclone probes from the chromosome region lp36 being used for the hybridization and a total of 87 PI clones being isolated in the process. Fluorescence in situ hybridization (FISH) was used to map 46 Pl clones on human metaphase chromosomes. Based on this extensive analysis, it was possible to assess the quality of the human PI library used (available from the Reference Library Database, ICRF, London, UK) in terms of complexity, size of the clone inserts and chimerism rate. The result of this analysis represents the prerequisite for the use of PI clones for processes in which labeled hybridization probes are made visible on chromosome sections and in particular in the interphase chromatin.

The present invention relates to a method for the investigation of chromosomal DNA by means of non-radioactive in situ hybridization. The method is characterized in that Pl clone DNA is used as the hybridization probe. The method is used in particular for genome analysis and for the diagnosis of diseases that can be attributed to chromosomal aberrations.

In principle, all known methods are suitable as methods in which nucleic acid probes which are provided with a non-radioactive label are made visible after attachment to the genomic DNA. Such methods are available in a large selection, an overview of such methods is e.g. the article by Lichter et al., 1991; reference is made to the original articles cited in this review article.

In a preferred embodiment, the method is used as fluorescence in situ hybridization. The principle of this method ("FISH") is to visualize DNA probes on chromosome sections using fluorescent dyes, either directly, with the hybridization probe itself carrying the fluorescent label, or indirectly, e.g. using immunological methods. Examples of embodiments of the FISH method are among others von Lichter et al., 1992.

In the context of the present invention, the efficient and simple use of PI clones as probes for fluorescence in situ hybridization with metaphase preparations and interphase nuclei could be demonstrated. The intensity of the fluorescence signals was very high, the hybridization efficiency of all clones evaluated was approximately 100%. In the context of the present invention, with regard to the saturation cloning of a limited genome region of 20 to 25 Mbp, a new method combination consisting of microcloning and a library of Pl clones was used. Another aspect of the present invention is thus a method for isolating as complete a number of clones as possible from a PI clone library from a defined chromosome section, in which microclones from this section are used as hybridization probes. The production of microclones has been described by Weith et al., 1989, among others.

Using 80 probes from an lp36-specific microclone library, hybridization screening was carried out on a large scale with a PI library which was cloned onto matrix filters in a high clone density. A detailed characterization of the isolated Pl clones, in particular by means of FISH analysis, provided comprehensive information about the usability of the Pl cloning system for both genome analysis and diagnostic methods. During the various hybridization series carried out in the context of the present invention, it was found that the hybridization signals on positive PI colonies were already clearly above the signal background after very short exposure times; the signals were also clear and unambiguous. (The results of the FISH analysis showed that all of the Pl clones tested mapped lp36 in the target region examined, indicating that no false positive clone had been isolated.) This distinguishes the Pl cloning system from the YAC system, whose Hybridization signals sometimes appear diffuse due to the longer length. The PI clones also have the advantage that the inserts grow during growth and the storage of the clones appear to be stable, ie do not delete or react. The studies performed did not reveal a chimeric clone. (SelbsH; if minor fragments of chimerism should occur when using final fragments of Pl inserts as probes, the Pl system would be superior to the YAC system in this regard, which has a significant chimerism rate in the lp36 region.)

The production of probes from Pl clone DNA for FISH turned out to be surprisingly simple. The fluorescence signals from directly nick-translated Pl clone DNAs were remarkably strong and comparable to those of alphoid DNA probes. Compared to the properties of signals from other types of clones, such as cosmids, the Pl clone signals proved to be much more intense and, using

Standard fluorescence microscopy, much easier to evaluate. A comparably high hybridization efficiency can only be achieved with Alu-PCR-amplified YAC clones (Lengauer et al., 1992a and 1992b); however, the use of PI clones is both easier and less time consuming.

The detection efficiency of Pl-FISH probes in the tests carried out was 84 to 97% in interphase nuclei; the interphase fluorescence signals were clearer than those of YAC clones, which often give spotty diffuse signals. A clear calculation of the number of signals per core could be carried out for most probes; this is another prerequisite for the diagnostic use of PI clones in FISH diagnostics. Due to its length, the signal obtained with PI clones is strong enough to be made visible without electronic aids. It can be evaluated with can be made with the naked eye in normal incident fluorescence. On the other hand, the clones are small enough to provide a limited signal. In any case, the amount of labeling is large enough to achieve a high detection efficiency equivalent to that of the alphoid DNA probes. This equivalence in efficiency gives the combination of Pl clones with centromeric, highly repetitive probes in multi-color FISH experiments a high value in cytogenetic diagnostics.

Because of these properties of PI probes, particularly the remarkable signal intensity, they are an ideal tool for interphase cytogenetics and are therefore valuable for clinical and prenatal diagnostics as well as for other diagnostic systems, e.g. for tumor interphase z togenetics.

In another aspect, the invention relates to kits for non-radioactive in situ hybridization, in particular for fluorescence in situ hybridization.

In a preferred embodiment, a FISH kit contains in each case separate containers a) one or more PI clones which hybridize with the sequence in question in the chromosome region to be examined and with one which can be detected directly or indirectly by means of fluorescence microscopy Marking, for example biotin, is (are); b) a reference probe provided with another marking, e.g. digoxigenin, e.g. alphoid DNA (the reference probe contains chromosome-specific repetitive DNA sequences, e.g. centromeric sequences) and thus delivers locus-specific signals with high efficiency; such DNA probes are usually used used to detect numerical chromosomal aberrations); c) optionally Competitor-DNA, such as Cotl-DNA (available from GIBCO-BRL, for example), which saturates the repetitive sequences present on the chromosome due to its composition of highly repetitive DNA fragments (this means that the labeled probes only signal specific chromosome sections receive); d) commercial hybridization reagents, such as SSC, formamide, dextran sulfate.

The method according to the invention and the kits suitable for carrying it out can be used for the diagnosis of all diseases caused by chromosomal aberrations. Examples include neuroblastoma (Weith et al., 1989), DiGeorge syndrome (Carey et al., 1992), malignant melanoma (Guan et al., 1992), Wilms' tumor (Riccardi et al., 1978), colon cancer (Baker et al., 1989; Leister et al., 1990), breast cancer (Devilee et al., 1991), hepatoma (Fletcher et al., 1991; Simon et al., 1991) and rhabdomyosarcoma (Biegel et al., 1991) .

The invention can also be used as a mapping tool to narrow down aberrations found using conventional cytogenetic methods or to search directly for an aberrant gene. Appropriate selection of Pl clone pairs, e.g. telomeric and centromeric too

Translocation breakpoints, for FISH on the aberrant karyotype, allows the breakpoint to be narrowed down to a genome section of minimal size. For example, probes specific for PAX-7, HSPG2, ENOl, CDC2L1 or D1Z2 can be used for the precise localization of neuroblastoma-specific translocations or deletions. Another advantageous application of the present invention is to use PI clones that span specific breakpoints directly. Such clones give a characteristic split signal in FISH analysis.

Figure overview

Fig. 1: Secondary hybridization screen of PI clones. Fig. 2: Size determination of PI clone inserts. Fig. 3: Fluorescence in situ hybridization

The invention is illustrated by the following examples.

example 1

Hybridization screening of Pl clones

In order to obtain a high density of DNA markers for the distal section of the short arm of chromosome 1, microdissection and microcloning of the chromosomal region lpter-p35 (Weith et al., 1989) and lpter-p36.2 (Weith et al ., 1989; the cell line of the designation KK 609, obtained from ECACC, was used) 3,500 La bda bacteriophage clones were produced. 80 non-overlapping DNA microclone probes (containing single copy DNA) with an average size of 2.3 kbp (range 0.3 to 9.0 kbp) were randomly selected from these libraries. 43 of the bacteriophage inserts had been subcloned into a Bluescript vector, the inserts of the remaining 37 clones were subjected to a polymerase chain reaction (PCR) using printers 5 'and 3' to the cloning site of the bacteriophage vector (Kuziel and Tucker, 1987). The probes were labeled with ^ P-άCTP using random priming (Feinberg and Vogelstein, 1983).

Based on the Southern hybridization signals of the individual probes, 10 pools, each consisting of 8 DNA probes, which provide similarly strong hybridization patterns, were put together in order to serve as hybridization probes for the PI matrix filters. To radioactively label the probes, 50 ng of each of the eight clone DNAs within a pool were labeled with 3 p_3cτP (Feinberg and Vogelstein, 1983) and each sample for 30 min at 68 ° C with, μg sheared vector DNA ( Size: 300 bp, bluescrip, stratagene) and, if necessary, ie if the signal was diffuse, with titrated amounts of cotl-DNA (BRL / Life Technologies), pre-competed. The samples were then pooled and hybridized according to standard procedures (Church and Gilbert, 1984). Washing after hybridization was carried out with 40 mM sodium phosphate, 0.1% SDS at 65 * C for 2 x 40 min. The filters were then exposed on an X-ray film (Kodak X-Omat AR) for various periods of time (4 hours at room temperature to 3 days at -70 ° C).

These "pools of eight" were hybridized as probes to the ICRF human PI library.

It was assumed that a human PI library was produced from the lymphoblastoid cell line GM1416B (Human Genetic Cell Repository, Camden, NJ) according to the principle described by Sternberg et al., 1990, and Pierce and Sternberg, 1992, among others Reference Library System of the Genome Analysis Laboratory, ICRF, is available (Zehetner and Lehrach, 1994). The The Pl library used consists of approx. 47,000 Pl clones, of which 41,400 were spotted in high density by means of robots using the method described by Hoheisl et al., 1993 on two 22 x 22 cm membranes. The 41,400 clones correspond approximately to one genome equivalent: Depending on the quality of the membranes, up to eight hybridizations can be carried out per filter. A total of five different filter sets were used for the present exemplary embodiments.

In the first screening, 152 positive Pl clones were identified. The number of positive signals per "pool of eight" fluctuated between 7 and 25 (Table 1). 133 (out of the 152) PI clones were subjected to a second screening (19 clones were discarded because they had problems with propagation).

For the second screening, individual clones of the "pools of eight" were hybridized to filters which contained lysed colonies from all of the PI clones which had been detected in the respective first screening. Those positive PI clones found from each screen by hybridizing the "pools of eight" were obtained as single colonies from the ICRF and each clone separately in LB medium containing 25 μg / ml kanamycin at 37 * C cultivated. Aliquots of 0.5 μl per individual colony were spotted on rows of small membranes (GreenScreen plus) and grown overnight on agar containing LB and kanamycin at 37 ° C. After binding the colony DNA to the membrane by means of denaturation and renaturation, carried out according to standard instructions As described by Sambrook et al., 1989, the individual filters were hybridized with microclone probes from the respective hybridization pool in order to obtain the corresponding Detect Pl clones. The DNA of those PI clones that were positive in this secondary screen was prepared using a modified version of the alkaline lysis protocol (Pierce and Sternberg, 1992).

Fig. 1 shows the autoradiograms of 6 (out of a total of 8) microclone probe hybridizations of pool No. 10. The microclone probe of the designation pl-06 did not identify a single Pl clone (Fig. 1f), nor the probes of the designation pl-07 and pl-08 (not shown in the figure). The probes pl-01 and pl-02 each identified two Pl clones (Fig. La, b), while the probes pl-03 and pl-04 identified only one clone (Fig. 1c, d). Hybridization of the microclone probe pl-05 gave seven positive Pl clones (Fig. Le). Two (out of 15) PI clones that had been positive in the first screening could not be detected by any of the eight microclone probes in the second screening.

A total of 36 Pl clones (27%) that gave positive signals on the first screen could not be confirmed as positive on the second screen. 97 of the 133 Pl clones examined were finally confirmed and identified by 41 individual microclone probes (Table 1). These 41 DNA probes detected between 1 and 7 Pl clones (on average 2.4 Pl clones per DNA probe), while no corresponding Pl clones could be found with the other 39 DNA probes. The second screen also showed that ten of the 97 Pl clones were detected twice by probes from different pools, reducing the total number of positives to 87.

Example 2 Size determination of the Pl clone inserts

For the size determination of the cloned inserts, the DNA from 50 Pl clones with 20 units of Notl was digested for 3 h at 37'C and through

Field inversion gel electrophoresis (FIG. 1% agarose gel at 200 V, 161 mA, 1) for 12 seconds forward, 4 seconds backward for 8 hours; 2) 6 seconds forward, 2 seconds backward 13 hours; 3) 3 seconds forward, 1 second backward 3 hours) fractionated. The result of the fractionation is shown in Fig. 2: Lane A: Lamda ladder (New England Biolabs); Lane B: high molecular weight marker (Gibco BRL / Life Technologies). The analyzed Pl clones showed an average insert size of 73 kbp. In fact, the size was between 22 kbp and 95 kbp, but only 5 clones of Insertt were shorter than 50 kbp.

Example 3

Fluorescence in situ hybridization (FISH)

In this example, the position of the Pl clones in lp36 was checked using FISH and the chimerism rate was determined. At least one PI clone per individual microclone was analyzed; in this way as much information as possible about the positive clones was obtained. 46 clones were mapped using FISH analysis of the prometaphase chromosomes.

The following methods were used (unless otherwise stated, the manufacturer's instructions were followed): i) Marking the probes for FISH

The Pl clone DNA was labeled with Bio-11-dUTP (Sigma) or dig-11-dUTP (Boehringer Mannheim) by nick translation (Lichter et al., 1992). The plasmid probe pucl.77, which contains the pericentromeric region of human chromosome 1 (Cooke and Hindley, 1979), was nick-translated with dig-11-dUTP.

ii) Human metaphase preparations and interphase nuclei

Human metaphase preparations and interphase nuclei were prepared from phytohaemagglutinin-stimulated normal human blood lymphocytes, from the lymphoblastoid cell line KK 609 (obtained from the ECACC) and from bone marrow biopsy material from neuroblastoma patients. Standard techniques (colcemide treatment, hypotonic treatment, methanol / acetic acid fixation and slide coating, as described by Cremer et al., 1988) were used. The preparations were stored in 70% ethanol at 4 * C. Before use, the slides were pretreated with RNase A (100 μg / ml) in 2xSSC for 60 min at 37'C, followed by a pepsin digestion (50 μg / ml) in 0.01 M HC1 for 10 min at 37 ° C, and one Post-fixation step in 1% acid-free formaldehyde in lxPBS / 50 mM MgCl2 for 10 min at room temperature (Wiegant et al., 1991).

iii) hybridization

Hybridization and detection of the probes were performed as described by Lichter et al., 1992, with the following modifications: 50 to 100 ng of labeled Pl clone DNA together with various amounts (1 to 5 µg) of an unlabeled Cot1 DNA fractions were dissolved in 10 ul 50% formamide, 10% dextran sulfate and 2xSSC. The DNA mixture was denatured for 5 min at 75 ° C. and incubated for 30 min at 37 ° C. for pre-annealing. For the two-color FISH experiments, the hybridization mixture was labeled with 5 ng as reference probe and denatured (75 ° C. during 5 min) Pericentromer probe DNA (dissolved in 50% formamide, 10% dextran sulfate and 2xSSC) was added to the slide immediately before the task The cells were denatured at 73 ° C. in 50% formamide 2xSSC (pH 7.0) Hybridization took place at 37 "C for 15 h.

iv) Immunological detection

The slides were washed 3 times in 50% formamide, 2xSSC at 45 * C, followed by three washes with O.lxSSC at 60 * C. Then the slides were pre-incubated for 30 min at 37 "C in 4xSSC, 5% bovine serum albumin, followed by three layers of immunological detection reagents: 1 .: avidin-FITC (VECTOR), 2 .: biotinylated goat anti-avidin (VECTOR), 3 .: Avidin-FITC. Between the changes, the excess antibodies were removed by three washing steps (37 * C, 5 min) in the same buffer as for the immunological detection (4xSSC, 0.1% Tween 20). Biotinylated probes were analyzed with avidin (vector) conjugated with fluorescein isothiocyanate (FITC) The FITC signals were amplified 1x (Pinkel et al., 1988). Digoxigenin-labeled probes were isolated by indirect immunofluorescence using mouse anti-digoxin (Sigma) and rabbit anti -Mouse IgG-TRITC (Sigma) Detected after detection of the signal, the cells were counter-stained with 0.1 μg / ml 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were fluorescent-anti-fading- Buffer (1 mg p-phenylenediamine in 1 ml glycerol buffer, pH 8.0) (Lichter et al., 1992).

v) Conventional and digital fluorescence microscopy

The signals were evaluated with a Zeiss Axiophot epifluorescence microscope equipped with a 100 W mercury lamp. A cooled CCD camera (Photometrics, Tuscon, USA), equipped with a Kodak KAF 1400 chip, was used on the microscope for digital fluorescence microscopy. The software package Nu200 2.0 (Photometrics) was used to obtain a digital image conversion in "TIFF" format. The black and white fluorescence images were taken using the Zeiss filter set 01 (BP 365, FT 395, LP 397) for the DAPI signals, filter set 15 (BP 546, FT 580, LP 590) for the rhodamine signals and filter set 09 (BP 450-490, FT 510, LP 515-565) recorded for the FITC signals. After the format change from TIFF to PICT, the further processing of the images, including overlaying and representation in false colors, was carried out with the software package “Gene Join”, as described by Ried et al., 1992.

a) All 46 Pl clones mapped in the chromosome region lp36 for the FISH analysis of metaphase preparations from normal human lymphocytes. None of these clones produced further signals anywhere else in the genome; this suggests that all clones tested are non-chimeric. (In fact, three Pl phages showed hybridization signals on the telomeres of different chromosomes, including the lp telomer. However, since all three of these clones were identified using the same microclone probe assumed that the complex, telomer-associated hybridization patterns are due to cross-hybridization of conserved telomeric or subtelomeric sequences that are also present on other chromosomes. ) After FISH analysis of the 46 biotinylated PI clones on 50 metaphases from normal human lymphocytes, it was found that all metaphases showed the expected signals on both chromatids of each of the homologous chromosomes; these signals were already visible with standard incident fluorescence without using the CCD camera.

b) In addition, the efficiency of fluorescence in situ hybridization with PI clones was examined with regard to hybridization with interphase nuclei in order to test the suitability of this type of clone for clinically applicable cytogenetic diagnoses. For this purpose, seven randomly selected PI clones were biotinylated or labeled with digoxygenin-11-dUTP and hybridized with both normal human lymphocyte nuclei and nuclei from a lymphoblastoid cell line. The biotinylated probes were detected with FITC, those with digoxygenin with TRITC. The number of fluorescence signals obtained in the interphase core was based on the theoretically expected number of signals. This evaluation was facilitated by the clear, clear signals and the lack of background fluorescence due to the sufficient suppression of repetitive sequences. The data obtained are summarized in Table 2. The expected number of two signals per core could even be detected in 84 to 97% of the examined cores without a CCD camera and digital image analysis.

c) Since alphoid DNA probes are generally considered to be the most efficient FISH probes, the Hybridization efficiency of the Pl clones compared with this type of probe. A Pl clone, specific for lp36, and the repetitive probe pucl.77, which labels the pericentromeric region of chromosome 1, were simultaneously hybridized with nuclei from human bone marrow cells.

Figure 3 shows representative examples of FISH analysis. The signals recorded with a CCD camera were processed using digital image analysis and displayed in false colors. In the black-and-white image, DAPI appears as a diffuse area and within it the FITC signals as small white spots and the TRITC markings (in FIG. 3b) as slightly larger dark gray to black, brightly delimited spots). The R-band pattern obtained, which is due to counterstaining of the chromosomes with DAPI, indicates the mapping position of the PI clone in lp36.2-3. 3a: Part of a metaphase preparation from normal human lymphocytes after FISH of a biotinylated PI clone which had been isolated using an lp36-specific microclone probe. The hybridization signals were clearly limited to lp36 and could be detected on both chromatids of the two chromosomes.

3b: Interphase nuclei of bone marrow cells after two-color FISH of a biotinylated PI clone and the digoxygenin-11-dUTP-labeled probe pucl.77. In all nuclei, two distinct yellow signals, which represent the PI clone, and two more extensive red signals, which extend over the pericentromeric region of the chromosome, were detected in all nuclei. The figures show that the fluorescence signals from the Pl clones were generally of remarkable intensity and specificity; they were comparable to that IS

Signals as obtained with alphoid DNA probes.

Isolation of Pl clones by hybridization screening of 80 microclone probes from the chromosome region lp36

Pool positive stable positive positive clones per clone propagated clones of individual microclone probes eight 1st screen bare clones 2nd screen No. pl p2 p3 p4 p5 p6 p7 p8

1 15 14 12 2 2 l ^a 7

2 21 17 12 1 4 2 2b 2 1

3 25 19 14 5 1 3 1 1

0) σ

4 19 18 11 ₄ c 3 1 1 ro

5 12 12 9 c 3 ^d + l 1 ^ro

6 11 9 6 2 ^b +2 2

7 7 6 4 1 1

8 11 9 6 2 1 * 3 <*

9 14 14 10 3 2 2

10 17 15 13 2 2 1

Total 152 133 97 ad: Superscript letters indicate identical PI clones.

Table 2

Calculation of FISH signals in interphase cores

Mark N

1 bio - 2 97 1 - 103

2 bio - 3 94 3 - 320

3 dig 2 6 84 6 2 100

4 bio - 1 94 4 1 107

5 dig - 1 92 7 - 109

6 bio 4 8 86 2 - 250

7a bio 1 4 92 1 2

434

7b dig - 2 95 2 1

bio: biotin dig: digoxigenin

Lines 1-3: lymphocytes

Lines 4-5: lymphoblastoid cell line

Lines 6-7: bone marrow cells

Lines 1-6: different PI clones were hybridized individually and calculated

Lines 7a, b: Two-color FISH analysis of a biotinylated Pl clone and a digoxigenin-labeled pucl.77 probe a) calculation of the Pl signals b) calculation of the pucl.77 signals from the same nuclei as in a)

N: Number of cores evaluated literature

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Claims

2 patent claims

1. A method for examining chromosomal DNA, in particular chromosome aberrations, by means of non-radioactive in situ hybridization, characterized in that Pl clone DNA is used as the hybridization probe.

2. The method according to claim 1, characterized in that the PI clone DNA has a label which gives a detectable signal by means of fluorescence microscopy.

3. The method according to claim 2, characterized in that the labeling of the Pl clone DNA is indirectly detectable.

4. The method according to claim 2 or 3, characterized in that, together with the Pl-DNA probe, a reference probe is used which contains alphoid sequences and which has a label which is different from the labeling of the Pl-clone DNA in the fluorescence microscope Signal there.

5. Kit for fluorescence in situ hybridization, comprising, in addition to conventional hybridization reagents in separate containers a) one or more Pl clones provided with a label which can be detected directly or indirectly by means of fluorescence microscopy, the one or those to be investigated with the sequence in question Hybridize chromosome region; optionally b) a reference probe provided with a differently detectable marking in the fluorescence microscope; optionally c) a competitor DNA containing highly repetitive DNA sequences. Use of Pl clone DNA probes for the diagnosis of diseases in mammals, in particular humans, which can be attributed to chromosomal aberrations by means of fluorescence in situ hybridization.