EP2321432A1

EP2321432A1 - Method for detecting chromosomal abnormalities

Info

Publication number: EP2321432A1
Application number: EP09740385A
Authority: EP
Inventors: Pierrick Serge Romana; Marc Le Lorc'h; Héra DER-SARKISSIAN
Original assignee: Assistance Publique Hopitaux de Paris APHP; Universite Paris 5 Rene Descartes
Current assignee: Assistance Publique Hopitaux de Paris APHP; Universite Paris 5 Rene Descartes
Priority date: 2008-07-25
Filing date: 2009-07-24
Publication date: 2011-05-18
Also published as: IL210846A0; FR2934279A1; WO2010010312A1; CA2731969A1; US20110207123A1

Abstract

The invention relates to an in vitro method for detecting chromosomal abnormalities in a mammal, including the in situ hybridization of n chromosomes from a metaphase chromosome preparation with sets having a plurality of nucleic acids, each set being hybridized, over a length of 700,000 to 3,000,000 contiguous bp, in a manner specific to the subtelomeric ends specific to said chromosomes, each set being detectably marked by a fluorochrome such that each chromosome is distinguishable by a particular fluorochrome or a particular fluorochrome combination.

Description

METHOD FOR DETECTING CHROMOSOMAL ABNORMALITIES

The invention relates to the detection of chromosomal abnormalities by hybridization of specific nucleic acid probes.

Technological background:

Chromosomal abnormalities, whether constitutional or acquired, are at the root of many pathologies. They can be balanced (that is to say, not accompanied by loss of chromosomal material) or unbalanced, that is to say accompanied by losses or gains in chromosome material. We are talking about genomic imbalances. With the techniques currently available routinely (band karyotype and fluorescent in situ hybridization), it is estimated that chromosomal abnormalities are responsible for 10 to 15% of mental retardation and congenital malformations. Moreover, they are very often found associated in cancer cells. In many cases, they determine the prognosis, and they can understand the molecular cause and consider targeted treatments. That is why, whether in constitutional pathology or in cancer, the diagnosis of chromosomal abnormalities is fundamental for both treatment and research. Chromosomal abnormalities may be visible under the microscope or not. When they are, their detection is provided by conventional karyotype techniques. Developed in the 1960s, they make it possible to visualize the chromosomes with a succession, along their longitudinal axis, of clear and darker bands characteristic of each pair of chromosomes. Examination of the global morphology of the chromosomes and the ordering of the different bands makes it possible to establish the karyotype. The smallest observable band is 5 million base pairs (5 megabases or Mb). This is a first limit to this technique. A second is that exchanges of telomeric regions, of the same size and whose hue (generally black) is identical, can not be detected by conventional karyotype techniques. These anomalies not detectable by the band techniques are called cryptic.

An approach to detect these cryptic chromosomal abnormalities implements specific fluorescent probes that hybridize on chromosomes. This technique, called FISH for "fluorescence in situ hybridization" (FISH), consists in hybridizing on the target DNA a complementary DNA fragment into which a fluorochrome has been introduced (so-called labeling operation). Such a DNA fragment is called a probe. Fluorescence in situ hybridization can be carried out on metaphase chromosomes, in which case it uses probes specific for a chromosome, an arm, a chromosomal region, a given locus. Chromosomal abnormalities are revealed by microscopic visualization of the fluorescent sites corresponding to the target DNA sites hybridized with the probes.

One of the major indications of FISH is the detection of telomeric cryptic abnormalities. These anomalies alone cause about 5% of mental retardation with malformations. Moreover, in cancers, telomeric abnormalities (balanced in these pathologies) have been described, the frequency of which is important. Thus, the t (12; 21) acute leukemia of the child is invisible by conventional cytogenetic techniques whereas it is the most frequent genetic abnormality in this pathology (Romana et al, Genes Chromosomes Cancer 1994, 9 (3): 186-91). Currently commercially available chromosomal anomaly detection systems are composed of one or two probes representing the telomeric ends of one or two chromosomal pairs. This supposes, if one wants to study all the ends, to hybridize these probes on one or more blades, by depositing them on different parts of these blades. This system assumes chromosome preparations rich in metaphases so that mitoses are present on all parts of the slide where the different probes will be deposited. These prerequisites can not be achieved for chromosome preparations from amniocyte cultures in prenatal constitutional cytogenetics or in cancers. For these two situations, it remained necessary to develop a system allowing the study of all chromosome ends on one or two mitoses.

This is what Brown et al (Nature Medicine, 7 (4): 497-501) wanted to answer by developing the M-TeI system. In this system, the telomeric probes of a chromosome are labeled with a specific combination of fluorochrome. Thus, they could make probes specific for 12 chromosomes, each detectable after hybridization on a single metaphase. Using two sets of probes, all 23 chromosome pairs could be explored by studying two mitoses. This test, subsequently used in the Brown et al study, 2002, Blood, 99 (7): 2526-2531, nevertheless has certain disadvantages. In particular, the probes used produce a weak signal with a signal / noise ratio that is far too high.

There was still a need for a cryptic telomeric anomaly detection test that was quick, convenient, and reliable. The test proposed by the inventors meets these requirements and is also usable not only for the diagnosis of prenatal and postnatal constitutional chromosomal pathologies, but also for the diagnosis of those associated with cancers. This test is also particularly suitable for the detection of balanced chromosomal abnormalities.

Summary of the invention

The invention provides an in vitro method for detecting chromosomal abnormalities in an animal, preferably a vertebrate, more preferably a mammal, comprising:

the in situ hybridization of n chromosomes of a metaphase chromosome preparation with sets of a plurality of nucleic acids, each set hybridizing over a length of 700,000 to 3,000,000 bp, specifically at specific subtelomeric ends said chromosomes, each set being detectably labeled with a fluorochrome, such that each chromosome is distinguishable by a particular fluorochrome or a particular combination of fluorochromes, n being an integer between 2 and the total number of chromosomes of said animal the total number of fluorochromes used being an integer less than n,

the detection of fluorescence emitted by the fluorochromes, whereby chromosomal anomalies are detected.

Legend of the Figure:

The Figure shows a general protocol for obtaining DNA from telomeric clones. Detailed description of the invention

Definitions:

A "specific subtelomeric end" is the specific region of a chromosome closest to the telomeric consensus sequence of a chromosome (common to all chromosomes). It is generally between 100 to 300 kb consensus sequences.

A "telomere" or "telomeric end" is a highly repetitive, non-coding region of DNA at the end of a chromosome. In humans, the TTAGGG sequence is telomere-specific and is repeated several hundred times. This sequence is common to all chromosomes.

By "specific hybridization" is meant the ability of a set of a plurality of nucleotides to hybridize to a given DNA sequence. In other words, a set that hybridises specifically to a subtelomeric end of one chromosome does not hybridize to another subtelomeric end. For example, a complex that hybridises specifically to the subtelomeric end of the long arm of chromosome 1 will not hybridize to the subtelomeric end of the short arm of chromosome 1, nor to any other sequence of this chromosome or chromosome. another chromosome.

For the sake of convenience, what is hereinafter called "target chromosome" is the chromosome suspected of being affected by an abnormality.

Sample Preparation Specimens to be tested are preparations of biological fluids or tissues containing metaphase chromosomes of an animal. Preferably these are blood samples, amniotic fluid in the case of prenatal diagnosis or bone marrow for hematological malignancies. Preferably the animal is a mammal, preferably a human, irrespective of sex or age. It can be an embryo, a fetus, a newborn, a child, a teenager, or an adult.

The samples are prepared as for a conventional cytogenetic slide analysis, whether samples taken in culture or analyzed directly. A general procedure for preparing metaphase chromosomes is to culture, if possible, the cells containing the chromosomes to be analyzed and to stop them in mitosis with a mitotic spindle inhibitor, for example with colchicine. The chromosomes are then fixed with an acetic acid / methanol mixture and then spread on a slide.

Probes used:

The method of the invention utilizes nucleic acid probes that span a length of 700,000 to 3,000,000 bases (bp) contiguous with at least one subtelomeric end of each target chromosome. Preferably, the probes cover at least 800,000 bp, more preferably at least 1 Mb, more preferably 1 to 1.5 Mb, subtelomeric ends specific for chromosomes.

The probes are constituted by a set of a plurality of nucleic acids (hereinafter "a set") covering all or parts of the specific subtelomeric end of the long arm or the short arm of the target chromosome. The plurality of nucleic acids is composed of nucleic acids complementary to contiguous portions of the subtelomeric end, all of which are specific. These contiguous parts may possibly be slightly overlapping. The subtelomeric end to which the probes hybridize is not that of a short arm of an acrocentric chromosome (i.e. chromosomes 13,14,15,21 and 22 in humans). As shown in Table 2, sets of nucleic acids hybridize at a variable distance from the telomere, depending on the chromosome targeted. For example, the nucleic acid arrays used in the examples hybridize at a distance of 4110 bp from the proximal end of the telomere 21q, or 828,698 bp from the proximal end of the telomer Ip. This distance is related to the requirement of specificity of the probe. Indeed, some even subtelomeric regions of a given chromosome may have common DNA sequences with other chromosomes. The size of these shared sequences may be variable depending on the chromosomes. This explains the chromosome variability of the distance between the telomeric consensus sequence and the distal end of the specific subtelomeric probe.

The total number of chromosomes present in the normal cells of a given animal is fixed and known. Thus, the cells of a human being normally comprise 23 pairs of chromosomes, one pair of which corresponds to the XX or XY chromosomes. In the method of the invention, n chromosomes are analyzed simultaneously, n being an integer between 2 and the total number of chromosomes of the animal. Preferably, at least 12 chromosomes are analyzed simultaneously.

According to a preferred embodiment of the invention, the set of a plurality of nucleic acids is produced by amplification of one or more BACs in which is inserted a fragment of the subtelomeric end of the target chromosome. Different websites allow to visualize the position of these BACs along each chromosome. For example, on the UCSC website (http://genome.ucsc.edu/)

The amplification can be carried out by any means of DNA amplification known to those skilled in the art. As an example, mention may be made of PCR amplification or amplification of the Rolling Circle Amplification (RCA) type.

Marking of the probes:

In the method of the invention, each set is detectably labeled with a fluorochrome, so that each chromosome is distinguishable from other chromosomes by a particular fluorochrome or a particular combination of fluorochrome.

The nucleic acids composing the set of a plurality of nucleic acids can be labeled with nucleotides conjugated with a fluorescent molecule, or fluorochrome

(direct marking).

The incorporation of nucleotides conjugated with a fluorochrome in the probes of the invention may be carried out by any means known to those skilled in the art, in particular by nick-translation or PCR. Alternatively, the nucleic acids can be labeled with nucleotides conjugated with a molecule specifically recognized by a ligand, preferably an antibody conjugated to a fluorochrome (indirect labeling).

More particularly, the indirect labeling of the probes used for the in situ hybridization of the target chromosome is carried out by a coupling of each of them to a hapten, and by an affinity reaction between this hapten and a ligand able to bind specifically. to said hapten and which is either conjugated to a fluorochrome, or rendered fluorescent by reaction with a fluorochrome-conjugated counter-ligand. As examples of haptens that may be used for indirect labeling of the probes useful in the present invention, mention may notably be made of:

digoxigenin, dinitrophenol and 2-acetylaminofluorene, in which case the hapten / ligand affinity reaction is advantageously carried out by means of antibodies directed against these compounds and conjugated to a fluorochrome;

biotin, in which case the hapten / ligand affinity reaction is advantageously carried out by means of fluorochrome conjugated avidin, or by using anti-biotin antibodies, then antibodies directed against them and which are conjugated to a fluorochrome .

The fluorochromes used may be indifferently chosen from various fluorescent compounds used for labeling nucleic probes such as fluorescein (sodium fluoresceinate) and its derivatives such as fluorescein isothiocyanate (FITC); rhodamine and its derivatives such as tetramethyl rhodamine isothiocyanate (TRITC); diaminidophenyl indo (DAPI); acridine; reactive amine fluorescent dyes such as succinimidyl ester of 6 - ((725 ami-4-methylcoumnarin-3-acetyl) amino) hexanoic acid (A. NICA); fluorescent dyes sold under the trade names BODIPY such as BODIPY'J, FR-Br, BODIPY R6G, BODIPY TMR, BODIPY TR and BODIPY 530/550 sold by Bio-Rad Inc. (USA), Cascade Blue dyes (Trilink BioTechlnologies USA), Cy2, Cy3. Cy3.5. CYS, Cy5.5, and Cy7 (Bio-Rad Inc., USA), DABCYL and EDANS (Eurogentec, BE); Eosin; Erythrosin; 6-Farn and Texas Red. Coumarin and its derivative diethyl aminomethyl coumarin (DEAC) are also preferred fluorochromes.

The process according to the invention is a multi-fluorescence process. In other words, the number of fluorochromes used for the detection of chromosomal abnormalities is less than the number n of chromosomes analyzed. Chromosomes are therefore marked in a combinatorial manner to distinguish them from each other. This chromosome labeling is performed by each set of a plurality of nucleic acids. Preferably, the subtelomeric ends of the short and long arms (p and q, respectively) of the same chromosome are labeled with the same fluorochrome or the same combination of fluorochromes.

In a preferred embodiment, at least four fluorochromes are used (for example direct labeling by FITC fluorochromes, rhodamine, coumarin, and indirect biotin, the latter being revealed by streptavidin labeled with Cy5), allowing 15 different combinations of fluorochromes (April ² -l). To detect abnormalities on all the chromosomes of an animal with more than 15 chromosomes, we can then use two batches of probes (for 12 chromosomes for one and 11 chromosomes for the other for the study of human chromosomes , for a total of 41 probes that hybridize all the ends of the long and short arms of human chromosomes, except the short arms of acrocentric chromosomes). Thus, from the analysis of a mitosis chosen on two different zones of a slide on which the two batches of probes have been deposited, all the telomeric ends may be studied. Preferably, the batches of probes are grouped so as to avoid analyzing in the same group cytogenetically-like chromosomes (for example, human chromosomes 21 and 22 are preferably analyzed by 2 different batches).

Table 1 below theoretically illustrates the 15 possible combinations for 4 different fluorochromes:

Table 1

Hybridization:

Hybridization is carried out according to the standard conditions of in situ hybridization (Romana et al., 1994, supra); As an indication, the metaphase chromosomes suspended in a mixture of acetic acid / methanol (1 / 3-2 / 3) are spread on slides. After being dried in air, they are immersed for 5 minutes in 2XSSC saline solution, dehydrated in alcohol solutions of increasing concentration (60% to 100%) and air dried. The two batches of probe are then denatured for 10 minutes at 70 ^° C. in 50% Formamide (FLUKA) / 2XSSC (Standard Sodium Citrate) at pH 7. The denatured probes are deposited on the slide at two different locations and covered by two lamellae. hermetically sealed with a "rubber cernent" glue. The set is put on a hot plate for 3 to 4 minutes. The whole is then hybridized for 18 hours at 37 ° C. in a humid chamber. Post-hybridization washes are performed at 72 ° C for 5 minutes in IXSSC buffer. In the case of using probes labeled with digoxigenin or biotin in particular, the detection of the probes is by the use of antibodies coupled to different fluorochromes.

In the case of using probes directly labeled with fluorochromes, the chromosomes can be counter-stained with DAPI (1 μg / ml, Molecular Probes) and mounted in p-phenyl-diamine. . Variations of these procedures are presented in the experimental section.

Visualization:

The revelation is generally after 18 hours of incubation at 37 ° C.

The slides are observed using an epifluorescence microscope equipped with a CCD camera and appropriate filters. The images are analyzed using a computer image processing system.

A chromosomal anomaly will be detected if there is no signal at a subtelomeric end of a target chromosome, or if there is a signal that is not located at the chromosome corresponding to the probe. Applications:

The method of the invention can be implemented for the detection of two or more chromosomes or, preferably, for all the chromosomes of the chromosome preparation. The method of the invention is useful not only for the diagnosis of constitutional chromosomal pathologies, but also for the diagnosis of cancers. This test is also particularly suitable for the detection of balanced chromosomal abnormalities. It is particularly useful in routine diagnosis, for the verification of chromosomal abnormalities suspected by chromosome examination by banding techniques. Moreover, it can be used for the diagnosis of cryptic chromosomal anomalies in prenatal and prenatal constitutional chromosomal pathology and in cancers, especially in malignant hematological malignancies (where they are often balanced).

The hybridization zone between the subtelomeric end analyzed and the set of a plurality of nucleic acids which is specific to it allows the detection of a high intensity signal. This has the main advantage of increasing the signal-to-noise ratio.

In addition, the combinatorial fluorescent labeling is particularly advantageous if the quantity of samples available for diagnosis is restricted. Indeed, as indicated above, the use of a combination of 4 fluorochromes allows analysis on two mitoses.

The examples and figures illustrate the invention without limiting its scope.

EXAMPLES

Example 1 Construction of 41 Telomeric Probes

The probes are prepared from BACs (Bacterial Artificial Chromosome). A BAC is a human DNA fragment, typically between 150,000 and 200,000 base pairs in length (150kb-200kb), inserted into a plasmid transfected into E. coli.

Different websites allow to visualize the position of these BACs along each chromosome. By consulting the UCSC website (http://genome.ucsc.edu/), the inventors selected 286 BACs located in the subtelomeric regions specific to the long and short arms of all chromosomes, with the exception of those of the short arms. acrocentric chromosomes (13, 14, 15, 21 and 22) which are composed of repetitive sequences common to these 5 chromosomes. For the 41 ends (23 chromosomal pairs minus 5), the BACs were chosen contiguously over a distance of at least 800,000 base pairs (800 kb) to obtain a probe generating a strong signal.

Table 2: characteristics of the probes used (dist of the telo = distance of the telomere)

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1.1. Culture of BACs

The BACs are ordered from the National Sequencing Center in Evry (Génoscope), which supplies them in the form of colonies in petri dishes.

Each BAC is cultured according to the following protocol: 500 μl of TB culture medium containing chloramphenicol (25 μg / ml) is deposited in each of the wells of the 96-well plate. Then one colony of each bacterial clone is cultured per well. The plates are incubated for 24 hours at 37 ° C. with shaking. The cultures are stopped by adding 500 μl of TB medium / 30% of glycerol. This will form the Glycerol Stock plate, from which 3 replicas will be generated: the M plate (Mother), the S plate (Backup) and the T plate (Work) each containing 150 μl of culture. The plate T is used to obtain the DNA. The plates are stored at -80 ^° C. in different freezers.

1.2. Amplification of DNA by RCA (Rolling Circle Amplification): This is the stage of DNA production a) Principle

DNA amplification by RCA is based on constant temperature replication of short single-stranded circular DNA segments. The reaction consists in the extension of randomly bound hexameric primers to the DNA template to obtain linear single-stranded DNA copies, which can in turn serve as a template for hybridization of other primers. This avoids the conventional phenol-chloroform extraction steps.

b) Protocol: GE Healthcare Europe Kit: ref 25-6400-80

This step uses the protocol provided with the RCA kit. It is carried out in plate 96 which allows the simultaneous production of DNA (10 μg) from 4 μl of culture of 96 different clones.

A final DNA concentration of 100 ng / μl (10 μg in 100 μl of water) is obtained. The verification of the amplification is done on agarose gel.

1.3. Marking

This is the step that will allow the incorporation of fluorescent molecules into the DNA.

Each product of RCA is marked by the principle of nick-translation.

In 2.5 hours, a solution containing 20 ng / μl of DNA labeled with a fluorochrome is obtained. The markings are checked as follows:

4 ml of the labeling reaction are deposited on 1% agarose gel in order to visualize under UV, the DNA in which the fluorochromes have been integrated. This allows to appreciate the quality of the marking.

1.4. Precipitation of the probes

This step allows the manufacture of the ready-to-use probe.

200ng of each of the probes are precipitated in the presence of 5OX (ie 10 μg) of Cotl DNA (DNA rich in repeated sequences which will block the existing repeat sequences in the probe), with 0.15M of 3M NaCl and 3 volumes of ethanol 100 %. The pellet is then taken up in 6 μl of hybridization buffer (50% formamide, 2 × SSC, 0.1% SDS, 40 mM NaH 2 PO 4 / NaHPO 4). The concentration of use of the probe is therefore about 30ng / μl.

1.5. Checking the location of the probes by FISH

Before constituting a probe contig, each probe (BACs) was verified by FISH on metaphase. All BACs generating a signal on several chromosomes, or not in place on the expected chromosome, have been replaced.

1.6. Formation of the "pool"

This makes it possible to simplify the process considerably by manipulating only 41 DNA pools in place of the 286 clones constituting it.

The "pool" plate is formed from the work plate. For each chromosome end, 20 μl of TB / glycerol, each bacterial clone corresponding to a BAC constituting the contig, are removed and then mixed in a single well of a plate 96. This consists of a plate containing 41 mixtures of bacterial clones from of which are produced very rapidly, according to the methods described above (culture, RCA, labeling, and verification by FISH), probes specific to each of the 41 chromosome ends.

From plate T (20μl of each BAC)

I ¹ PI! Q I ² PI ² q I 3p I 3q I ⁴ PI ⁴ q I ⁵ PI ⁵ q I 6p I 6q I

Plate 41 ends = plate "pool"

Example 2: Manufacture of the MTP (Mega Telomeric Probes) System

The strategy adopted led to the manufacture of 41 probes that are labeled by a combination of 5 fluorochromes: FITC, rhodamine, DEAC, biotin (revealed by streptavidin-Cy5) and digoxygenin (revealed in Cy5.5). The ends of the same chromosome are marked by the same color combination.

2.1 Marking of the two groups

Each group was constructed to avoid collecting cytogenetically similar chromosomes (21 and 22, in two different groups, for example).

All ends that contain the same fluorochrome are marked together by nick-translation and according to Tables 3A and 3B below. For group I, 1960ng of DNA were labeled in FITC, 2120ng in rhodamine, 1050ng in coumarin, 1983ng in Biotin, and 1860ng in digoxygenine in volumes of 83, 98, 50, 88 and 84μl, respectively. For group II, 1810ng were labeled in FITC, 1860ng in Rhodamine, 1607ng in Biotin, and 1944ng in digoxygenine. This difference in the amount of labeled DNA is intended to enhance the signal of some weaker ends. Tables 3A and 3B: Marking tables of the two groups with combinatorial table A- B-

2.2. Precipitation of groups

For hybridization and by group, all the labeled probes are precipitated simultaneously and taken up in 6 .mu.l of hybridization buffer.

2.3. Hybridization of probes Hybridization of so-called "Mega telomeric probes" probes (MTP) is carried out according to the same protocol as for the BACs, but requires two-depot slides to simultaneously treat the two groups of chromosomes.

2.4. Blade revelation and analysis

The slides are washed in an IXSSC bath at 72 ° C for 5 min and then revealed by two layers of antibody to reveal the biotin labeled probes and digoxygenin. The first layer of antibodies (50 μl of sodium bicarbonate IX + 2 μl of anti streptavidin Cy5

(lmg / ml) for biotin + 0.5μl of mouse anti-digoxigenin and the second layer (50 μl of 1X sodium bicarbonate + 1μl of anti Cy5.5 mouse (lmg / ml)) allows the revelation of digoxygenine .

Slides are mounted with 10μl of DAPI / Vectashield® solution (Vector

Laboratories) then covered with a 24x60 coverslip.

The slides are observed using an epi-fluorescence microscope equipped with a camera and appropriate filters and the metaphases analyzed using an analysis system. Example 3 Demonstration of a Balanced Subtelomeric Translocation t (5; 11) (q35; _P 15)

To test the resolution of the probes, the inventors hybridized them in a patient with acute myeloid leukemia associated with a cryptic telomere abnormality, the t (5; l I) (q35; pl5) invisible therefore in classical cytogenetics .

This translocation recombines the NUP98 genes encoding a 98 kDa nucleoporin located at 3Mb of the short arm telomer of chromosome 11 and the NSD1 gene encoding the nuclear receptor SET domain protein located at 4 Mb from the long arm telomer of chromosome 5. The Karyotype resolution in bands is 5 to 10 Mb (often 10Mb for chromosomes of leukemic cells). The MTP system of the invention has made it easy to detect this anomaly.

To confirm that this is indeed a translocation remodeling the distal end of the long arm of a chromosome 5 and that of the short arm of a chromosome 11, the inventors have hybridized the specific probes of the short and long arms of the chromosome 11 (labeled with coumarin (blue) and biotin with Cy5 streptavidin (yellow) and short (labeled with FITC, green) and long arms (labeled with Rhodamine, red) of chromosome 5. The inventors have thus been able to visualize a translocation t (5; 11) (qter; pter).

Claims

An in vitro method for detecting chromosomal abnormalities in an animal, comprising: - in situ hybridization of n chromosomes of a metaphase chromosome preparation with sets of a plurality of nucleic acids, each hybridizing set, on a length of 700,000 to 3,000,000 bp contiguous, specifically to specific subtelomeric ends of said chromosomes, each set being detectably labeled with a fluorochrome, such that each chromosome is distinguishable by a particular fluorochrome or a particular combination of fluorochromes where n is an integer from 2 to the total number of chromosomes of said animal, the total number of fluorochromes used being an integer less than n,

2. Method according to claim 1, the method being implemented for the detection of at least twelve chromosomes.

3. Method according to any one of claims 1 or 2, the method being implemented for all the chromosomes of the preparation of metaphase chromosomes.

The method according to any one of claims 1 to 3, wherein the sets of a plurality of nucleic acids is produced by amplification of one or more BACs into which are inserted one or more fragments of the subtelomeric end of said chromosome.

The method of claim 4, wherein the amplification is a Rolling-Circle Amplification (RCA) type amplification.

The method of any one of claims 1 to 5, wherein the nucleic acids of at least one set are labeled with nucleotides conjugated with a molecule specifically recognized by a fluorochrome labeled ligand.

The method of any one of claims 1 to 6, wherein the nucleic acids of at least one set are labeled with nucleotides conjugated with a fluorochrome.

The method according to any one of claims 1 to 7, wherein for each chromosome, the subtelomeric ends of the short arm and the long arm are labeled with the same fluorochrome or with the same combination of fluorochromes.

The method of any one of claims 1 to 8, wherein the animal is human.

The method of any one of claims 1 to 9 for diagnosing a constitutional chromosomal pathology.

11. Method according to any one of claims 1 to 9 for the diagnosis of cancer.