WO2001029253A2 - Simultaneous visualization of eu- and heterochromatin - Google Patents

Abstract

The present invention relates to a method for the visualization of eu- and heterochromatin comprising hybridizing to a target DNA a repeat-depleted probe and a probe specific for repetitive sequences. Further, the present invention relates to the use of said probes for the visualization of eu- and heterochromatin, as well as compositions, i.e., diagnostic compositions, and kits comprising said probes.

Description

Simultaneous visualization of eu- and heterochromatin

The present invention relates to a method for the visualization of eu- and heterochromatin comprising hybridizing to a target DNA a repeat-depleted probe and a probe specific for repetitive sequences. Further, the present invention relates to the use of said probes for the visualization of eu- and heterochromatin as well as compositions, i.e., diagnostic compositions, and kits comprising said probes.

Traditional FISH experiments use either a complex DNA probe for a euchromatic region (e.g. painting probe, chromosome region specific microdissected probe, YACs, PACs, cosmids, etc.) or a repetitive probe which hybridizes specifically to a certain region (e.g. chromosome specific centromere probe) or to several loci in the genome (e.g. the [TTAGGG]_n sequence at the telomeres). Euchromatic and heterochromatic probes are hybridized under different hybridization conditions. Euchromatic probes contain beside single copy sequences also repetitive elements, such as Alu- or L1 repeats which occur ubiquitous in the genome. In order to hybridize the euchromatic DNA probes specifically, the repetitive elements have to be suppressed. Traditionally, the specific hybridization of complex probes is achieved by the addition of excess quantities of an unlabeled blocking agent (or "competitor" DNA), such as human genomic DNA or Cot-1 DNA (Landegent et al. 1987; Lichter et al. 1988; Pinkel et al. 1988). The addition of Cot-1 DNA, however, hampers the simultaneous use of repetitive and euchromatic probes. Because these probes have to compete with the Cot-1 DNA they are either completely suppressed or yield at best very weak signals. The only reliable option to hybridize both probe sets simultaneously is the separate preparation of both probe entities in different tubes in order to denature them under different conditions and to mix them quickly immediately prior to the hybridization on a slide. This procedure is cumbersome, requires skilled and trained personnel, and is not feasible in routine laboratories. The major hurdle are the repetitive elements within the complex probes and therefore different procedure were developed within the last few years to deplete repetitive elements or to use other probe sources. Thus, to avoid the use of a blocking agent, a number of different approaches, listed below, were developed: One approach achieved specific hybridization of a limited number of probes by extended self-preannealing prior to hybridization (Wienberg et al. 1997). Recently, protocols based on affinity chromatography and positive (Chen-Liu et al. 1995; Rouquier et al. 1995) and negative (Craig et al. 1997) subtraction hybridization were developed to generate FISH probes depleted of repetitive sequences. The approach described by Craig et al. (1997) consists of three steps. In the first step, biotin-labeled "subtractor" DNA is hybridized to a source DNA in solution. Next, subtractor-source hybrids are separated from nonhybridized source DNA molecules using affinity chromatography. Then, the nonhybridized source DNA is purified and amplified by PCR, and the quality of depletion is tested in a FISH experiment. If the desired degree of selection has not yet been achieved, further rounds of depletion can be performed, either with the same subtractor DNA again or with a different DNA source as a subtractor.

A potential new probe source for the painting of human chromosomes without suppression could be chromosome paints derived from hominoid primates (Mϋller et al. 1997). Here, no blocking agent is needed because during evolution repetitive sequences diverged at a higher rate than single-copy sequences (Warburton and Willard 1996). However, a complete set of these probes to paint all human chromosomes is not yet available.

Furthermore, probe sets for the complete analysis of individual chromosomes are not available. Traditionally, only the euchromatin is analyzed. Therefore, structural rearrangements such as polymorphisms cannot be identified and in case of complex rearrangements (e.g. in tumor metaphase spreads) no conclusions about the origin of heterochromatin are possible. Normal FISH-analysis cannot visualizate the complete genome in a hybridization. Traditional M-FISH analyzes the euchromatin, reverse M-FISH the heterochromatin. Thus, it is not possible to collect as many data from the genome in one hybridzation as possible. Thus, an easy and cheap method to facilitate the analysis of genomic DNA should find widespread applications.

Thus, the technical problem underlying the present invention is to provide means and methods which allow the simultaneous visualization of eu- and heterochromatin.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a method for the visualization of eu- and heterochromatin comprising hybridizing to a target DNA a repeat-depleted probe and a probe specific for repetitive sequences.

Surprisingly, it was found that the depletion of repetitive elements from euchromatin DNA-probes allows the hybridization of euchromatin probes and of repetitive probes under similar conditions. As a result, the repeat-depleted probes can be mixed with, for example, complex euchromatin and/or heterochromatin probes, in one hybridization step or the hybridization of both set of probes can be performed subsequently under similar conditions. This will allow a simultaneous visualization of eu- and heterochromatin.

The term "euchromatin" as used herein refers to all regions of the genome in the interphase nucleus except for the heterochromatin.

The term "heterochromatin" as used herein refers to regions in the genome, that are permanently in a high condensed condition and are not genetically expressed. Usually "heterochromatin" comprises a high rate of repetitive sequences. The term "hybridizing" in accordance with the present invention denotes the pairing of two polynucleotide strands by hydrogen bonding between complementary nucleotides. By "hybridizing" it is further meant that a nucleic acid molecule hybridizes under certain hybridization conditions. The used stringency has to be adjusted for different probe sets. Particularly, in in situ hybridizations some probes require, e.g., "normal" stringency conditions (viz. 50% formamide, 10-20% dextransulfate, washing with 3x5 min each 4xSSC/0.5% Tween 20 at 45°C and 1xSSC at 60°C (e.g. rd-painting probes, alphoid probes, satellite III, rDNA, pUCI .77, [TTAGGGjπ, Yqh, 1 p36) while others (e.g. satellite I and the β-satellite probe) need very low stringency conditions. For example, for low stringency conditions, one hundred nanograms of the labeled probe DNA and 10 ug of salmon sperm DNA in 30% deionized formamide, 2xSSC, and 10% dextran sulfate can be denatured by heating to 75°C for 5 min and applied to the slide under a cover glass. The hybridization is carried out at 37°C overnight in a sealed moist chamber. For example, for low-stringency washing conditions the slides are washing three times for 5 min in a 30%, 2xSSC wash solution prewarmed to 45°C and three times for 5 min in 2xSSC at room temperature. Non-specific signal can be then blocked by incubation in 4xSSC and 3%BSA for 30 min at 37°C. It is also possible, to denaturate chromosomes for 2-3 min in 70% formamide, 2xSSC at 80°C, and then to dehydrate in an ethanol series (70%, 90% and 100%). The hybridization solution, e.g., contains 2ng/μl probe, 50 μg/μl salmon sperm DNA, 50 μg/ml yeast RNA, 30% formamide, 4xSSC, 50mM NaH₂P0₄/Na₂HPO , pH 7.0, 1 mM EDTA. The hybridization is then performed at, e.g., 35°C for 14 h, and the slides are washed three times for 5 min in 2xSSC at room temperature. An example of stringent hybridization condition in regard of a Southern blot is hybridization at 4xSSC at 65°C, followed by a washing in O.lxSSC, 0.1%) SDS at 65°C for one hour Sambrook (Molecular Cloning; A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Alternatively, an exemplary stringent Southern blot hybridization condition is hybridization in 50% formamide, 4xSSC at 42°C. Examples of non- stringent hybridization conditions are 4xSSC at 50°C or hybridization with 30-40% formamide at 42°C. Low stringency washing conditions are for example washing in 1 x SSC or in 37°C in 5 x SSC at 50°C.

The term "a repeat-depleted probe" as used herein relates to a probe or probes which has/have a low amount of repetitive sequences. Euchromatic probes are usually produced by performing an amplification reaction, e.g., a PCR, of a eukaryotic genomic DNA sequence comprising template DNA, e.g., a chromosome, a YAC or total genomic DNA. Eukaryotic genomic DNA sequences contain often a high percentage of repetitive sequences as mentioned above. Accordingly, eukaryotic probes are complex DNA probes containing beside single copy sequences also repetitive elements such as, e.g., Alu or L1 -repeats. Particularly, most methods used to generate euchromatin probes, such as interspersed repetitive sequence PCR, produce probes which comprise a high percentage of repetitive sequences. Further methods as well as templates for the production of euchromatin probes are well known in the art.

Under "repeat-depleted probe" it is here understood that said probe contains less repetitive sequences than the original eukaryotic DNA, e.g., the chromosome or chromosomal region, which served as template for its production. The production of a repeat-depleted probe might be performed in one step or via one or more intermediates in more than one step. The intermediates can have the same, a reduced or increased percentage of repetitive sequences compared to the probe. Accordingly, "a repeat-depleted probe" contains at least 5% less repetitive sequences than the template which was used for its production. Preferred is the reduction of about 10% or more, more preferred is a reduction of about 25% or more, even more preferred is 50% or more reduction. Most preferred is a reduction of the repetitive sequences in the probe of more than 70%>. Preferred is a reduction of repetitive sequences to such an extent that the probe yields specific hybridization signals under the hybridization conditions as exemplified herein. The probe depleted of repetitive elements can be produced according to protocols well known in literature. Several approaches for the generation of repeat-depleted DNA are discussed above and are included herein by reference, i.e., Reulin et al. 1995, Rouquier et al., 1995, Craig et al., 1997, Mϋller et al., 1997, Warburton and Willard, 1996. With "repeat-depleted probe" it is also meant that a probe contains repetitive sequences which are not contained in said target DNA. For example, probes from hominoid primates can be used in accordance with the present invention if the target DNA is a different DNA, for example, human DNA. During evolution the repetitive sequences of primates diverged from those of e.g. humans. Therefore, said probes do not need to be combined with any blocking agent. Example 2 describes the generation of repeat-depleted probes by a selective hybridization method. Furthermore, it was found that due to the much faster hybridization kinetics of repetitive sequences repetitive DNA fragments hybridize faster compared to single copy sequences under appropriate PCR conditions. This effect causes that repetitive elements are already double stranded by the time the polymerase reaction starts. As a result, single copy DNA can be preferentially amplified and repeat-depleted DNA can be obtained under said appropriate PCR conditions. Accordingly, the generation of repeat-depleted DNA can be achieved by a first PCR, wherein the hybridization step is a low stringency hybridization step, and a second PCR following the first PCR, wherein the hybridization step of said second PCR is a high stringency hybridization step; see Example 7.

As used herein, the term "probe" means a probe or probes and relates to polynucleotides or polynucleotide composition(s), and which are used to identify another polynucleotide by hybridizing therewith. Accordingly a probe is/are (a) labeled polynucleotide(s) which means that it/they can be visualized by methods well known in the art. A probe might be DNA or RNA. For example, a previously repeat- depleted DNA can be transcribed into RNA, which then can be used as probe (if appropriate). The method of the present invention relates to any combination of probes as long as at least one repeat-depleted probe and one probe specific for repetitive sequences is used. The person skilled in the art knows to vary the probe set according to the requirements of the actual application. Accordingly, differently labeled probes, e.g. probes labeled with different dyes, e.g., for M-FISH, and/or probe set specifically developed for a specific application might be used. The term "probe" comprises primers, PCR products, vectors, cosmids and other polynucleotides which can be labeled. Different procedures allow the efficient labeling of probes. The labeling procedure itself is not important for a successful depletion of repetitive sequences. Such labeling methods of polynucleotides are well- known to a person skilled in the art (e.g., Sambrook, 1989). For example, DNA is labeled by random priming or nick translation. Further labeling techniques comprise the introduction of modified bases or nucleotides into an amplification reaction, e.g., the addition of biotinylated residues, fluorescence residues or radionucleotides to a PCR or primer extension or labeling can be obtained by e.g. the Biotin Chem-Link method (Boehringer, Mannheim). A labeled repeat-depleted probe can be used as a probe for hybridizing or for screening, e.g., in situ hybridizations, polynucleotide arrays or polynucleotide blots, e.g. a DNA or RNA array, or a Southern or Northern blot. Polynucleotide arrays (or microarrays) can be applied e.g. for expression analysis, genotyping, or mutation screening. A state of the art overview about applications and techniques is provided in Nature genetics supplement, 21 (1999), 2- 30, which is incorporated herewith by reference.

The term "repetitive sequence" relates to sequences, which occur many times within a genome. Those sequences are found mainly in heterochromatic DNA. Studies of the kinetics of the re-association of formerly denatured DNA revealed that eukaryotic

DNA, in contrast to prokaryotic DNA, contains many repeated base sequences. The most highly repetitive DNA in the human genome is the so-called satellite DNA

(Cotι/2 value as low as 10^"3), which is composed of a very long series of tandem repetitions of a short nucleotide sequence. Satellite DNA is often found in the heterochromatin associated with the centromere chromosomes, where it may contain several thousands homologous repeat units.

Highly repetitive satellite DNA is also interspersed (singly or in tandem arrays) throughout the genome. Historically, the interspersed repeats have been grouped into two classes: the short interspersed nuclear elements (SINES), consisting of repeats shorter than 500 bp, and the long interspersed nuclear elements (LINES).

The Alu family of genetic elements is 300 bp SINE, with about 900,000 copies (3 to

6% of the genome) dispersed throughout the genome, giving an average distance between copies of about 4 kb. The Alu sequence is beiieved to have become dispersed throughout the genome by a translocation mechanism that is partially encoded within the element. The L1 genetic element is a LINE sequence found at about 20,000 to 50,000 per genome, but consists of elements averaging 6 to 7 kb in size. A detailed overview about human repetitive elements was recently published by

Lee et al. (1997).

Following, a list of repetitive probes examples with their localization within the genome is provided: α-satellite: A 171 bp monomer, present at all centromeres.

Alphoid chromosome-centromere specific α-satellite probes (synonym: α-satellite probes): for almost all chromosomes as, e.g., 1 , 1 +5+19, 2, 3, 4, 4+9, 6, 7, 8, 9, 10,

11 , 12, 13+21 , 14+22, 15, 16, 17, 18, 20, 22, X, Y; alphoid chromosome-centromere specific α-satellite probes: for almost all chromosomes generated by alphoid PCR

(Dunham et al. 1992); β-satellite: This is a 86 bp monomer probe, locating the centromeres of chromosomes 1 , 9, at Yq and hybridizing to all acrocentric chromosomes.

Some special repetitive probes, specific for certain regions within the genome: 1 p36

(specific for heterochromatic block at the distal arm of 1 p); pUC1.77 (Sat III, 1q12);

D15Z1 (Sat III; 15p1 1.2); pHUR-195 (Sat II; variable heterochromatic region of chromosome 16); Yq (Sat III; variable heterochromatin of the long arm of the Y chromosome; microdissected probe); Telomere probe rTTAGGG]_π: specific for all telomeres. rDNA: Four pools of human ribosomal DNA (viz. 5.7 kb, 6.4 kb, 11.9 kb, and 19.8 kb EcoRI fragments; Labella & Schlessinger 1989) are used for the delineation of rDNA. The EcoRI inserts are cloned into pUC9 and amplified and labeled by DOP-PCR. Satellite DNA family 1 (predominant family of simple repeats of the classical satellite I family): 42 bp repeat, arranged as alternating 17 bp and 25 bp repeat units. Localized at the pericentromeric regions of chromosomes 3 and 4 and all acrocentrics. Satellite DNA family 3 (predominant family of simple repeats of the classical satellite III family): 5 bp (ATTCC) repeat, occasionally interspersed with the specific 10-bp sequence (A^T _GTCGGGTTG). Localized at the variable heterochromatin at 1q12, 9q12 and Yqh and all acrocentrics proximal to the rDNA regions and in close proximity to α-satellite DNA of chromosomes 10 and Y, probably also at chromosomes 5, 17, and 20.

Other repetitive sequence families are the gamma satellite family, the 48-bp satellite DNA family, the Sn5 satellite DNA family, short interspersed repeated elements (SINEs; could be generated by Alu-bands, long interspersed repeated elements (LINEs), the 724 sequence family, the Long Sau DNA family, AT-rich sequence DNA, the chAB4 multisequence family.

The term "target DNA" relates to any DNA comprising eu- and/or heterochromatin. Therefore, the method of the present invention can be used for the visualization of eu- and/or heterochromatin from many different origins. Thus, target DNA can be, e.g., chromosomes, i.e. metaphase chromosomes, yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), cosmids, vectors or plasmids. The DNA can be isolated from cell lines, e.g. hybrid cell lines, isolated by microdissection or generated by other PCR protocols. Furthermore, under "target DNA" total or partial genomic DNA, or DNA derived from plastids, i.e. chloroplast DNA or mitochondria DNA is understood. Specific chromosomes might be isolated, e.g. by microdissection, and then be used as target. Accordingly, in a preferred embodiment the hybridizing of the repeat-depleted probe and the probe specific for repetitive sequences occurs under the same hybridization conditions.

The term "same hybridization conditions" as used herein relates to hybridization conditions of identical or similar stringency for each probe. Accordingly, the visualization of hetero- and/or euchromatin can be achieved under identical hybridization conditions. Furthermore, it is understood that said probes are not hybridized necessarily in one hybridization solution. The term "same hybridization conditions" also relates to hybridizing first the repeat-depleted probe and subsequently the probe specific for repetitive sequences or vice versa in different hybridization solutions but under the same conditions and without removing the first probe. It might be that the hybridization conditions might vary slightly if a probe is hybridized to the target DNA subsequently. The stringency of the hybridization conditions depend as mentioned above form several factors, e.g., percentage of dextransulfate, percentage of formamide and/or posthybridization washing conditions. Different stringency strategies are shown above and in the enclosed examples.

In a preferred embodiment the repeat-depleted probe and the probe specific for repetitive sequences are hybridized simultaneously. The term "simultaneously" as used herein relates to a hybridization procedure, wherein a target DNA is incubated with said DNA probes in one hybridization solution.

In one embodiment the hybridization is an in situ hybridization. In a preferred embodiment the hybridization is a fluorescence in situ hybridization (FISH). Said in situ hybridization comprises many different hybridization types, e.g. chromosome painting, reverse FISH, chromosome-specific hetero- and euchromatin- bar coding, acrocentric chromosome hybridization, M-FISH, reverse M-FISH, and complete M-FISH. Thus, the present invention can also be used to generate hybridization probes for mapping naturally occurring genomic sequences. The sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads, flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price (Blood Rev. 7 (1993), 127-134) and Trask (Trends Genet. 7 (1991), 149-154). The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma, (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f) and Meinke, Science 282 (1998), 662-682. Correlation between the location of the gene on a physical chromosomal map and a specific feature, e.g., a disease, may help delimit the region of DNA associated with this feature. The method of the present invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.

In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps or genetic analysis. For example a sequence tagged site based map of the human genome was recently published by the Whitehead-MIT Center for Genomic Research (Hudson, Science 270 (1995), 1945-1954) and a map of the plant genome by way of the Arabidopsis genome is available from http://qenome.wwz.Stanford.edu/cqi-bin/AtDB/nph-blast2atdb. Often the placement of a gene on the chromosome of another species may reveal associated marker even if the number or arm of a particular chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for interacting genes or gene defects by said gene discovery techniques. Once such gene has been crudely localized by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The method of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals. As mentioned above the target DNA can also be e.g. chromosomal DNA, plasmids, cosmids, YACs, PACs, BACs, microdissected DNA, DNA generated by other PCR protocols, genomic DNA, or plastid DNA and therefore, said probes can also hybridize therewith. Furthermore, in accordance with the method of the present invention the probes can also be used for hybridizing to polynucleotide blots and polynucleotide assays comprising said target DNA or polynucleotides derived thereof.

Accordingly, in one embodiment the repeat-depleted probe is a chromosome painting probe.

Further, in another embodiment the probe of the method of the present invention is a combination of probes.

Accordingly, the probe can comprise one or more polynucleotide(s), and said polynucleotides can have different sequences. For example, a probe can be generated by performing a PCR with a degenerated primer set. Such a probe comprises many different polynucleotides of different lengths depending of the complexity of the used template of said PCR. Probes specific for different sequences can be combined to obtain a new probe. Accordingly, probes hybridizing to different repetitive sequences can be combined to obtain a new probe. Examples for probes hybridizing to repetitive sequences are mentioned above. Combinations of probes, are provided in the enclosed examples. Accordingly, it is possible to combine several repeat-depleted probes or probes hybridizing to repetitive sequences or both types of probes to obtain the desired hybridization pattern, i.e., to visualize specific acrocentric chromosomes, a complete chromosomal set, a single chromosome, (a) specific chrmosomal region(s), or a combination of chromosomes. The person skilled in the art knows to select probes according to the desired visualization. Several applications and corresponding probes as well as the preferred stringency conditions are shown in the enclosed examples.

In another embodiment the target DNA comprises one or more individual chromosomes.

In a further embodiment the target DNA comprises a complete chromosomal set. According to the above said, the target DNA can be a single chromosome, which has been, e.g., isolated by microdissecting, as well as several chromosomes, e.g., acrocentric chromosomes. Further, the target DNA can comprise chromosomal DNA which has been cloned into YACs, PACs, BACs, cosmids, vectors or plasmids. Furthermore, the target DNA can comprise a complete genome, i.e. all chromosomes of an animal or a plant. Further examples of target DNA are mentioned above.

Accordingly, in another embodiment the probe is specific for one or more acrocentric chromosomes or for one or more chromosomes or chromosomal regions. Such a probe finds a widespread usage in the characterization of genetic polymorphisms, e.g., of supernumerary marker chromosomes or acrocentric chromosomes. Supernumerary marker chromosomes are observed about ten times more frequently in the mentally retarded population (Buckton et al. 1985). SMCs are small structurally abnormal chromosomes that occur in addition to the 46 normal chromosomes. Their incidence is reported to be as high as 0.3/1000 in the general population (Buckton et al. 1985). The vast majority of SMCs (ca. 80%) are derived from acrocentric chromosomes, in particular chromosome 15, and don't have detrimental effects for the patient (Friedrich and Nielsen 1974; Buckton et al. 1985). About one fifth of SMC are familial and are generally transmitted without detrimental effects. The remaining 80%) arise de novo and are associated with a marked maternal age effect. Thus, it is important to determine whether a SMC consists solely of heterochromatin which is not associated with adverse phenotypic effects or whether a SMC also contains some euchromatin. However, the origin of SMC can usually not be delineated from banding analysis alone. Therefore, many investigators have used FISH to decipher the chromosomal origin of SMCs (Crolla et al. 1997, 1998; Blennow et al. 1993, 1994, 1995; Callen et al. 1990, 1991 , 1992; Plattner et al. 1993a, 1993b; Rauch et al. 1992).

Relatively well-studied supernumerary chromosomes include the inv dup(15) [account for approximately 40% of marker chromosomes, have been reported in normal individuals, individuals with mental handicap and other anomalies], the "cat eye" marker [Ag-NOR staining shows usually two signals at either end of the marker chromosome, the typical bisatellited isodicentric chromosome contains copies of the 22pter->22q11.2 region (McDermid et al. 1986, Liehr et al. 1992, Mears et al. 1994)], the i(18p) [a small, metacentric chromosome, can be identified on the basis of its size and banding pattern], the der(22)t(11 ;22)(q23;q11.2) [the only recurrent, non- robertsonian constitutional translocation seen in humans], and the i(12p) [Pallister- Killian syndrome, tissue specific mosaicsm; extra, small metacentric chromosome]. Furthermore, the short arms of acrocentric chromosomes 13, 14, 15, 21 , and 22 show a range of morphology. They usually harbor a number of different classes of tandemly repeated DNA. Starting from the centromere which consists of α-satellites these different classes appear from proximal to distal in the order satellite III, β- satellite (both combined are the proximal short arm), rDNA (satellite stalk), β-satellite and satellite I (both combined form the satellite region/ Gravholt et al. 1992; for review see Lee et al. 1997). However, detailed analysis of these regions on DNA fibers which allows a higher resolution indicated that there may be significant variation in the sequence of these repetitive fragments. The tandemly repeated rDNAs, located in the satellite stalks (secondary constrictions/ Goodpasture et al. 1976) are specific chromosomal regions that form and maintain the nucleoli in interphase nuclei and are therefore termed nucleolus organizer regions (NORs). [Each nucleolar organizer corresponds to a cluster of tandemly repeated rRNA genes on one chromosome. The nucleolar organizer is the region of a chromosome carrying genes coding for rRNA.] Specific silver staining (Ag-NOR-staining) of the NORs is used to demonstrate their transcriptional activity (DA Miller et al. 1976; OJ Miller et al. 1976) and most individuals have four to seven per cell that are active (Varley 1977).

The short arms of the acrocentric chromosomes show a range of morphology, reflecting variation in three components of the short arm: the centromeric heterochromatin, the satellite stalk, and the satellite material. At one extreme, a short arm may seem to be absent; at the other extreme, it may be so long that a D- group chromosome is of C-group appearance, and a G-group chromosome has an F- group resemblence. Satellites vary widely in appearance: apparently absent, small or large, and single or double. Advantageously, the method of the present invention allows the diagnosis of more than 50%, preferably more than 65%, more preferably more than 80% of all SMCs in one experiment with assessment about existing euchromatin. In addition, most preferably all acrocentric p-arm polymorphisms can be accurately evaluated. In another embodiment, the target DNA is derived from animals or plants. The target DNA can be derived from higher or lower eukaryotes, e.g. of mammals (e.g. humans, rats, mice, rabbit, etc.) plants, fungi or protozoa. Examples for plants may be crop plants, e.g. corn, wheat, barley, rice, oil seed crops, tree species, vegetables, fruits, etc.

Accordingly, in another embodiment, the target DNA is derived from specific cells or tissues, e.g. tumors, oocytes, sperms, embryonic tissues or cells obtained by amniocentesis.

In another embodiment, the target DNA is stained. Since the discovery that appropriate staining results in a banded appearance of chromosomes by Zech and Caspersson (Caspersson et al. 1968; Caspersson et al. 1970) various banding methods of metaphase chromosomes have been used in pre- and postnatal diagnostic applications as standard techniques. Giemsa bands obtained by digesting the chromosomes with the proteolytic enzyme trypsin (GTG-bands) are the most widely used in clinical laboratories for routine chromosome analysis. GTG-banding can only achieve a resolution to the single band level, i.e. approximately 5-10 million base pairs. Beside GTG-banding there exist other, selective banding technologies. The C-banding technique produces selective staining of constitutive heterochromatin. These bands are located mostly at the centromeric regions of chromosomes. The G-11 -bands-technique involves a modification of Giemsa staining at alkaline pH. Metaphases show selectively stained regions in chromosomes 1 , 3, 5, 7, 9, 10, 20, and the Y chromosome. The centromeric, proximal short arm and the satellite regions of acrocentric chromosomes are variably stained, depending on the characteristics of individual chromosomes. The T-bands-staining preferential stains of telomeric regions.

By silver impregnation (Ag-NOR staining) one or more dotlike structures of varying sizes appear, and may extend beyond the NORs (nucleolus organizing region) to the space between the acrocentrics. NOR refers to a specialized region at the short arm of acrocentric chromosomes. It consists mainly of rRNA genes, and forms in the interphase nucleus the nucleolus. Although consisting of genes the NOR region is part of the repetitive sequences within the genome. Hybridization to this region is usually suppressed by the addition of Cot-1 DNA to the hybridization mix. NORs, stained by silver, are localized in the secondary constriction regions or so called stalks (not satellites) in the short arms of acrocentric chromosomes. The number of silver-stained NORs per metaphase may vary in different individuals, usually ranging between 5 and 10. Only the active NORs are impregnated by silver: the silver- stained regions in metaphase chromosomes represent the active NORs that have participated in the formation of nucleolus in the preceding interphase stage. The proteins associated with the transcriptional activity of ribosomal cistrons are responsible for silver impregnation.

Further, in order to check whether a markerchromosome is derived from acrocentric chromosomes (and thus a small satellited marker) metaphase spreads can be counterstained with DA/DAPI (Distamycin A/4'-6-diamidino-2-phenylindole) which stains preferentially secondary constriction regions of chromosomes 1 , 9, and 16; the proximal short arm of 15; and the distal long arm of the Y chromosome. In addition, the pericentric regions of some of the chromosomes, such as 4, 7, 10, 19 and other acrocentric chromosomes show fluorescence of various intensities. The marker chromosomes, which show brilliant fluorescence by DA/DAPI staining, originate from chromosome 15, whereas those without significant fluorescence might have originated from either chromosome 15 or others. DA/DAPI produces a brightly fluorescent signal on chromosome 15p, but not on any of the other acrocentric chromosomes. Therefore, any acrocentric that was positive for a signal with DA- DAPI could be identified as being derived from chromosome 15 (Ram S. Verma, Arvind Babu: Human Chromosomes, Principles and techniques, 2^nd Edition, McGraw- Hill, Inc; Chapter 3: Banding techniques, pp. 72-134; Chapter 4: Spezialized techniques, pp. 134-172). A person skilled in the art knows to combine staining techniques with further applications. For example, it is clear to the artisan that Giemsa staining can be performed before or after performing a FISH but that the Giemsa staining itself disturbs FISH. Therefore an artisan would destain the Giemsa stained DNA with Ethanol or Fixativ before performing a FISH. Advantageously, hybridization and visualization is performed before staining.

In a further embodiment the method of the present invention is used for e.g. simultaneous visualization of eu- and heterochromatin, for the painting of individual chromosomes, in situ hybridization, FISH, M-FISH, CoM-FlSH, or ReM-FISH. Accordingly, it can be used for the analysis of tumor metaphases or for the analysis of acrocentric chromosomes, e.g., human chromosomes 13, 14, 15, 21 and 22. Accordingly, the method of the present invention can be used in pre- or postnatal diagnostic.

The use of the method of the present invention depends on the probes hybridized to the target DNA. The person skilled in the art knows which probe set can be used for any of the mentioned applications. Examples for applications and corresponding probes can be found in the enclosed examples. The method of the present invention can, for example, be advantageously performed to analyse tumor metaphases. It might be that such an analysis and the resulting findings lead to the development of a new set of probes which then can advantageously be used according to the method of the present invention.

In another embodiment a combination of a repeat-depleted probe and a probe specific for repetitive sequences is used for the visualization of eu- and heterochromatin.

The term "combination" as used herein relates to the hybridization of two or more probes with a target DNA without removing any probe hybridized to the template before visualization. Accordingly, a second probe can be hybridized to a template DNA subsequently to a first probe under identical hybridization conditions and without removing the former hybridized probe. Nevertheless, the solution comprising said first probe might be removed after the corresponding hybridization step has been performed. Also, washing steps might have been performed before the addition of the second probe. A group of probes or all probes can also be hybridized together within one hybridization solution.

In another embodiment the present invention relates to the use of a repeat-depleted probe and a probe specific for repetitive sequences for the preparation of a composition. In a preferred embodiment, the present invention relates to a composition comprising a repeat-depleted probe and a probe specific for repetitive sequences. The term "composition", as used in accordance with the present invention, comprises at least the repeat-depleted probe and the probe specific for repetitive sequences and, optionally, further molecules, either alone or in combination, like, e.g., molecules which are capable of supporting hybridization. The composition may be in solid or liquid form or in form of (a) powder(s).

The composition of the present invention may also be a diagnostic composition. Such a composition may furthermore comprise at least one of the aforementioned DNA, vectors, arrays or blots etc. and, optionally, suitable means for detection. Said diagnostic compositions may be used for methods for determining differences in the chromosomal composition due to translocation, inversion, etc. among normal, carrier or affected individuals. Methods of detecting the presence of a polynucleotide comprises hybridization techniques are well known in the art. Advantageously, the diagnostic composition is used for the identification of polymorphism, structural and complex chromosomal rearrangement, for pre- or postnatal diagnostic, the identification of supernumerary marker chromosomes (SMC), visualization of arcocentric chromosomes, e.g. of the p-arms of arcocentric chromosomes, or tumor cytogenetics.

The composition of the present invention can be provided in parts or together, and each part, e.g. a specific probe or a target DNA, e.g. a control DNA can be packaged individually.

In another embodiment the present invention relates to a kit comprising instructions for performing the method of the present invention. Said kit can further comprise a repeat-depleted probe and a probe specific for repetitive sequences.

Further, in another embodiment the present invention relates to a kit comprising a repeat-depleted probe and a probe specific for repetitive sequences. Advantageously, the kit of the present invention further comprises, optionally, a target DNA, (a) reaction buffer(s) and/or storage solution(s). Parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. The kit of the present invention may be advantageously used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications referred to above, e.g. in diagnostic kits or as research tools. Additionally, the kit of the invention may contain means for detection suitable for scientific and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.

Several documents are cited throughout the text of the specification. Each of the documents cited herein including any manufacturer's specifications, instructions, etc. are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprising" will be understood to imply the inclusion of a stated integer or a step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The figures show:

Figure 1 Top row: Hybridization with repeat depleted painting probe for chromosome 1 (red), probe 1 p36 (yellow), specific for the repetitive sequence at the 1 p telomere region, a chromosome 1 specific centromere probe (p/5.1 ; green), and probe pUC1.77 (blue) specific for the heterochromatic 1q12 block. The top row illustrates the hybridization pattern to a normal chromosome 1.

Second and third row: Hybridization of the same probe set to aberrant chromosomes from a tumor metaphase spread (non-small cell lung cancer). In the 2^nd row is a deletion of a large part of the long arm visible. The chromosome in the 3^rd row consists of chromosome 1 euchromatin, however, the centromere must be from another chromosome as none of the used probes stains the centromere.

Fourth row: In this example was a probe for the heterochromatic block of the Y-chromosome (Yqh, yellow) added to the probe mix. The probe mix reveals the complex composition of the chromosome, which has (from top to bottom) the Yqh hetereochromatic block, a small band of chromosome 1 euchromatin, the chromosome 1 centromere, chromosome 1q12 heterochromatin block, and chromosome 1 euchromatin.

Figure 2 Repeat-depleted chromosome 13 (red) hybridized together with a centromere probe specific for both, chromosome 13 and 21 (arrowheads) centromeres.

The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be provided as a limitation of scope of the present invention.

Example 1 : Chromosome-specific Hetero- and Euchromatin-Bar codes (CHEBs)

Probe sets for the complete analysis of individual chromosomes are not available. Traditionally, only the euchromatin is analyzed. These probes allow the simultaneous evaluation of both the eu- and the heterochromatin. Structural rearrangements such as polymorphisms can easily be identified and in case of complex rearrangements (e.g. in tumor metaphase spreads) are conclusions about the origin of heterochromatin blocks possible.

Chromosomal bar codes (FISH based technology) are usually constructed from a number of region-specific probes (e.g. YAC clones). Each clone is labeled with a different fluor or fluor combination (combinatorial labeling strategy). The result are multiple bands along the length of a chromosome. Intrachromosomal rearrangements (inversions, small duplications or deletions) can easily be identified and breakpoints can easily be mapped.

The following provides a summary for the construction of a chromosome 1 specific multicolor bar code which was constructed using different YAC clones. Only YAC clones with a hybridization efficiency of >99% were selected for the bar codes. YAC clones HTY3222 and YRM2123 are half-YACs specific for the telomeric chromosome bands 1 p36.3 and 1q44, respectively (Vocero-Akbani et al. 1996). In addition, several YAC clones were selected from the CEPH-library (Bray-Ward et al. 1996). Their suitability for a bar code was checked by hybridizing two or three YACs simultaneously in different colors to normal metaphase spreads in order to determine their relative position to each other and to map the exact band position at a high resolution level. Based on these hybridization results seven YACs were selected in addition to the two above mentioned half-YACs. After Alu-PCR (Lengauer et al. 1992) YACs were labeled according to a labeling scheme. Advantages: accurate break-point mapping and easy identification of intrachromosomal rearrangements. However, the traditional bar codes cover also only euchromatic regions.

The following probe sets are described as illustrating the above. A probe set for chromosome 1 can consist of the following probes: 1 p36, chromosome 1 centromere, pUC1.77, and replete depleted painting probe, each labeled in a different color. Probe set for a "High resolution chromosomal bar code", e.g. for chromosome 1 : Construction of a chromosome 1 bar code consisting of multiple YAC-clones as mentioned above: YAC clones HTY3222 and YRM2123 are half-YACs specific for the telomeric chromosome bands 1 p36.3 and 1q44, respectively (Vocero-Akbani et al. 1996). In addition, several YAC clones were selected from the CEPH-library (Bray-Ward et al. 1996). Their suitability for a bar code was checked by hybridizing two or three YACs simultaneously in different colors to normal metaphase spreads in order to determine their relative position to each other and to map the exact band position at a high resolution level. Based on these hybridization results seven YACs were selected in addition to the two above mentioned half-YACs. After Alu-PCR (Lengauer et al. 1992) YACs were labeled according to a labeling scheme. Now, in addition to the YAC clones are also repetitive probes (chromosome 1 centromere, 1q12, 1 p36) included in the bar code.

A probe set for an individual acrocentric chromosome, e.g. chromosome 14, can consist of repeat depleted painting probe, centromere specific probe, and rDNA. A probe set for an individual acrocentric chromosome, e.g. chromosome 14, can consist of repeat depleted painting probe, centromere specific probe, and satellite III. A probe set for an individual acrocentric chromosome, e.g. chromosome 14, can consist of repeat depleted painting probe, centromere specific probe, satellite III, and rDNA. A probe set for an individual acrocentric chromosome, e.g. chromosome 14, can consist of repeat depleted painting probe, centromere specific probe, satellites I and III, rDNA, and β-satellite.

A probe set for chromosome 17, can consist of a repeat depleted painting probe and a chromosome specific centromere probe. The importance of such a relatively simple probe set is exemplified by a structural abnormal chromosome 17 which was observed. The abnormality could not be resolved by traditional methods alone. Said probe set demonstrated a polymorphism of the centromere of this chromosome. These polymorphisms of centromeres occur frequently on chromosomes 1 , 9, and 16, however, they are rare on chromosome 17 (and also on the other chromosomes). In this situation cytogeneticists feel uncomfortable to establish a definite diagnosis, in particular in prenatal diagnostic applications.

Example 2: Generation of repeat depleted probes

The following is based on the procedure described by Craig et al. 1997 and Bolzer et al. 1999. Additional probe sources are mentioned above.

DNA probes

Chromosome-specific painting probes were generated by microdissection (Guan et al. 1994) or flow-sorting.

Preparation of painting probes for negative selection and affinity chromatography In the first step, the original DNA was amplified using the degenerate oligonucleotide- primed PCR (DOP-PCR) technique (Telenius et al., 1992). About 100 ng of source DNA was amplified in a volume of 50 μl with 1 x Taq DNA polymerase buffer (50 mM KCI, 10 mM Tris-HCI [pH 8.3]; GIBCO BRL), 2 mM MgCI₂, 0.2 mM of each dNTP, 1.7 μM primer 6MW (5'-CCG ACT CGA GNN NNN NAT GTG G-3' [SEQ ID NO: 1]), and 5 U Taq DNA polymerase (GIBCO BRL). Cycling conditions were as follows: (1) 95°C for 5 min; (2) four low-stringency cycles of 95°C for 1 min, 31 °C for 1.5 min, and 72°C for 3 min; (3) 32 high-stringency cycles of 95°C for 1 min, 60°C for 1.5 min, and 72°C for 1 min, with the addition of 1 s per cycle to the extension time; and (4) a final extension of 5 min at 72°C. In the second step, the DOP-PCR amplification products were amplified with a more stringent version of DOP-PCR called CTA₄-PCR (Craig et al., 1997). About 100 ng of DOP-PCR products were amplified by the CTA₄-PCR technique according to published protocols (Craig et al., 1997). The conditions were essentially the same as for DOP-PCR. The only differences include the replacement of primer 6MW by the primer CTA DOP (5'-CTA CTA CTA CTA CCG ACT CGA G-3' [SEQ ID NO: 2]), annealing was performed at 53°C without a time extension, and the number of normal cycles was 36.

PCR products were purified using either Sephadex G50 columns or commercial BioGel P6 columns (BioRad). All PCR products were checked on a 1 % agarose gel. The CTA₄-PCR amplification products were used for the negative selection and affinity chromatography.

Labeling of Cot-1 DNA with biotin

Twenty-five micrograms of Cot-1 DNA (GIBCO BRL) were biotinylated using a Biotin- Chem-Link kit (Boehringer Mannheim) according to the manufacturer's instructions. (If needed, this reaction can be scaled up for larger DNA quantities [Craig et al., 1997].) Biotinylated reaction products were purified on Sephadex G50 or commercial BioGel P6 (BioRad) columns and are referred to here as b-Cot1-DNA. The concentration of b-Cot1-DNA in solution after biotinylation was 250 ng/μl.

Selective hybridization and affinity chromatography

The procedures for selective hybridization and affinity chromatography were modified from those described by Craig et al. (1997). Usually, at least two rounds of depletion were done.

In a first round of depletion, first, each painting probe was depleted with b-Cot1-DNA. About 200 ng CTA -PCR product starter DNA, the amount of a single painting probe, was combined with a 25-fold excess of subtractor DNA in 2 x SSC in a final volume of 30 μl. The probes were denatured for 10 min at 95°C, placed on ice for 5 min, and hybridized for approximately 4-5 h at 37°C. Second, affinity chromatography with streptavidin-magnetic beads (Boehringer Mannheim) was performed exactly as described by Craig et al. (1997). In brief, the beads were prepared according to the manufacturer's instructions and incubated together with the hybridized probe DNA for 30 min at 37°C with axial rotation on a Thermomixer (Eppendorf). Thereafter, a magnetic particle separator (Boehringer Mannheim) was applied, the beads were captured along the tube wall, and the supernatant was transferred into a new tube. This was repeated once with a new set of beads.

After the solution was purified and concentrated, a second round of depletion was done using the DNA produced in the first round as new starter DNA. Again a 25-fold excess of b-Cot1-DNA was added, and the DNA was denatured as above. Contrary to the first round, the second round of hybridization was performed overnight at 62°C. Affinity chromatography was then carried out in the same way as in the first depletion round.

The resulting probe solution was cleaned using Microspin columns (Genomed JETQUICK or Quiagen QIAquick) and eluted in 30 μl of 10 mM Tris-HCI (pH 8). These 30-μl aliquots contained the desired repeat-depleted DNA probe and were used in a CTA -PCR. In general, the PCR products could not be visualized on a gel due to the small amounts of DNA, the only visible change being the vanishing primer. Therefore, a second CTA₄-PCR with 9 μl of the first PCR product as DNA template was needed. Usually the DNA probes were already labeled at this step according to the PCR labeling protocol detailed below.

Additional rounds of depletion for probes showed an unsatisfactory degree of selection. Depleted probes were tested with FISH. If the desired degree of selection was not satisfactory after the second round of depletion, a different DNA probe was used as a subtractor for further rounds of depletion. This was necessary for chromosomes 1 , 3, 12, 14, 18, 19, 22, and X. For all of these chromosomes, except chromosome 22, one depletion was done with a DNA probe specific for all alphoid regions of the genome. This probe was generated by an alphoid PCR, as described elsewhere (Dunham et al., 1992), and designated α-gDNA. Using the alphoid PCR protocol, centromere-specific repetitive probes were generated for each of chromosomes 1 , 3, 12, 18, 19, and X and designated α-Chr.<number>-DNA. Additional specific depletion rounds were done with the following sources as subtractor DNA: pUC1.77 and α-Chr.1 -DNA (for chromosome 1 ); α-Chr.3-DNA (chromosome 3); α-Chr.12-DNA (chromosome 12); chromosome 14 specific painting probe (chromosome 14); α-Chr.18-DNA (chromosome 18); α-Chr.19-DNA (chromosome 19); painting probes for chromosomes 14 and 19 (chromosome 22); and α-Chr.X-DNA (X chromosome).

FISH

For fluorescence in situ hybridization (FISH), the most important factors that influence stringency are percentage of dextransulfate; percentage of formamide and posthybridization washing conditions. Under "normal" stringency conditions for traditional euchromatic probes the percentage of dextransulfate is in the range of 10- 20% in the hybridization mix. The addition of dextransulfate results in an increase of the signal intensity, but reduces also the specificity of a probe. For the hybridization of heterochromatic probes the percentage of dextransulfate is lower (<1 % or no dextransulfate at all). Hybridization of euchromatin is usually done in 50% formamide hybridization mix whereas for many heterochromatic probes formamide in the range of 60-65%) is being used. However, some heterochromatic probes, such as satellite- probes require very low formamide concentrations in the range of 30-35%. The standard washing steps after FISH with euchromatic probes consist of 3x5 min each 4xSSC/0.5% Tween 20 at 45°C and 1xSSC at 60°C. Some heterochromatic probes require washing at room temperature only with 2xSSC.

The stringency has to be adjusted for different new probe sets. Some probes require "normal" stringency conditions (viz. 50% formamide, 10-20%) dextransulfate, washing with 3x5 min each 4xSSC/0.5% Tween 20 at 45°C and 1xSSC at 60°C (e.g. rd- painting probes, alphoid probes, satellite III, rDNA, pUC1.77. [TTAGGG]_n, Yqh, 1 p36) while others (e.g. satellite I and the β-satellite probe) need very low stringency conditions. The different sets have to be constructed according to the stringency requirements.

Probe concentrations in experiments with a single painting probe or five probes was 10-20 ng DNA per probe per microliter of hybridization solution. A series of FISH experiments was done with various probe concentrations of probe pUC1.77, which is specific for the heterochromatin at band 1q12 on chromosome 1 (Cook and Hindley, 1979) and other probes for heterochromatic regions. Five micrograms of salmon testis DNA (Sigma) was added to all probe mixtures. The probe mixtures were precipitated with ethanol and resuspended in a hybridization solution containing 50% formamide, 2 x SSC, and 15% dextran sulfate. Probe mixtures containing depleted probes or the pUC1.77 probe were denatured but not reannealed and hybridized for one or two nights at 37°C to metaphase chromosome spreads.

After hybridization, the slides were washed three times with 2 x SSC, 0.1 %> SDS at 45°C and then three times with 0.1 x SSC, 0.1 % SDS at 60°C (or alternatively with 3x5 min each 4xSSC/0.5% Tween 20 at 45°C and 1xSSC at 60°C). In experiments with biotin- or digoxigenin-labeled DNA probes, unspecific binding sites were blocked with 3%> BSA in 4 x SSC, 0.2% Tween for 30 min in a moist chamber. Biotin- and digoxigenin-labeled DNA probes were detected with Avidin-Cy3.5 and anti- digoxigenin-Cy7 (or rabbit anti-estradiol and anti-rabbit Cy5.5; all Amersham Pharmacia Biotech), respectively. After final washes with 4 x SSC, 0.2% Tween at 45°C, slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted in phenylenediamine antifade medium.

Epifluorescence microscopy and image analysis

The following summarizes microscopy and image analysis used. The successful evaluation does not depend on the use of the equipment as listed below, but should in principle be possible with any instruments.

A motorized epifluorescence microscope (Leica DMRXA-RF8) equipped with an eight-position filter wheel and a Sensys CCD camera (Photometries; Kodak KAF 1400 chip) can be used for image acquisition. The specification of the filter set and details about the microscope were published elsewhere (Eils et al. 1998). Microscope and camera are controlled by the Leica QFISH software (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). M-FISH image processing was done using the Leica MCK image analysis package (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK) which is based on an adaptive region-oriented approach for spectral classification (Eils et al. 1998). M-FISH results can be displayed either as "true- colors" which are the result of overlaying the five source images without further image processing or as "classification-colors" which are generated by above mentioned classification algorithm (Eils et al. 1998). CGH experiments were evaluated with the Leica QCGH software package (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Gray scale images with region-specific probes were overlaid without further image processing using the Leica QFISH software package (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). For the bar code images contrast enhancement, pseudo-coloring, and overlaying was done in Adobe- Photoshop.

Example 3: Characterization of supernumerary marker chromosomes (SMC) and p-arm polymorphisms

Supernumerary marker chromosomes (SMC) are observed about ten times more frequently in the mentally retarded population (Buckton et al. 1985). Thus, it is important to determine whether a SMC consists solely of heterochromatin which is not associated with adverse phenotypic effects or whether a SMC also contains some euchromatin. However, the origin of SMC can usually not be delineated from banding analysis alone. Therefore, many investigators have used FISH to decipher the chromosomal origin of SMCs (Crolla et al. 1997, 1998; Blennow et al. 1993, 1994, 1995; Callen et al. 1990, 1991 , 1992; Plattner et al. 1993a, 1993b; Rauch et al. 1992).

The short arms of the acrocentric chromosomes show a range of morphology, reflecting variation in three components of the short arm: the centromeric heterochromatin, the satellite stalk, and the satellite material. At one extreme, a short arm may seem to be absent; at the other extreme, it may be so long that a D-group chromosome is of C-group appearance, and a G-group chromosome has an F-group resemblence. Satellites vary widely in appearance: apparently absent, small or large, and single or double.

Traditional methods for the characterization of SMC and p-arm polymorphism comprised above described silver impregnation (Ag-NOR staining), and DA/DAPI (Distamycin A/4'-6-diamidino-2-phenylindole), or microdissection and DOP-PCR, FISH (either only alphoid probes or only euchromatin).

Microdissection and DOP-PCR (also called reveres painting) has been used for the characterization of marker chromosomes (Mϋller-Navia et al. 1995). However, it depends on the skill and experience of the operator. In cases of acrocentric chromosomes signals upon reverse painting are often observed on all short arms of the acrocentric chromosomes and do not allow to pinpoint the origin of the centromere to a specific chromosome.

Assingments of chromosomes can be done per exclusion (e.g. additional hybridizations with CEP 15 (satellite III), CEP 15 (alpha satellite), and CEP 13/21 (alpha satellite) to slides of the patient showed no hybridization signals, thus, marker is most likely 14 or 22) or due to the actually observed signals (e.g. probe for chromosome 15).

Adventageously an improved probe set for the characterization of acrocentric chromosomes can be used according the above. The already above mentioned aspects regarding stringency conditions, including a) percentage of dextransulfate; b) percentage of formamide; and c) posthybridization washing conditions apply also for these probe sets. The satellite pool consists of a combination of probes for satellites I and III and β-satellite. Following, several set of probes are listed, which help to overcome the above mentioned disadvantages of the mentioned traditional methods. One probe set can consist of repeat depleted chromosome painting probes rd13, rd14, rd15, rd21 , and rd22 together with α-satellite-probes (13+21 , 14+22, 22, 15), "satellite-pool", and rDNA. Such a probe set has the advantage, that about 80 % of all SMCs can be diagnosed in one experiment with assessment about existing euchromatin. In addition, all acrocentric p-arm polymorphisms can be accurately evaluated.

In another set repeat depleted chromosome painting probes rd13, rd14, rd15, rd21 , and rd22 together with α-satellite-probes (13+21 , 14+22, 22, 15), sat III, and rDNA can be used.

Furthermore, it is possible to use repeat depleted chromosome painting probes rd13, rd14, rd15, rd21 , and rd22 together with α-satellite-probes (13+21 , 14+22, 22, 15), sat III, rDNA, and [TTAGGG]_n.

Another probe set might include the low stringency probes satellite I and the β- satellite probes (if possible): repeat depleted chromosome painting probes rd13, rd14, rd15, rd21 , and rd22 together with α-satellite-probes (13+21 , 14+22, 22, 15), satellite I, β-satellite probe, sat III, rDNA, and [TTAGGG]_π. However, these probe sets are only examples which should illustrate the present invention but do not limitate its scope. Example 4: Visualization of the p-arms of acrocentric chromosomes

It is impossible to visualize on the p-arms of acrocentric chromosomes the different satellite fractions (viz. β-satellite, satellite 1 , satellite 2, satellite 3) due to resolution limitations. However, the resolution of the different satellite fractions is irrelevant for diagnostic applications. Therefore a "satellite-pool", consisting of above mentioned fractions should be generated. On the p-arms of acrocentric chromosomes this should generate a sequence of signals α-satellite-"satellite-pool"-rDNA-"satellite- pool".

Pools of human ribosomal DNA EcoRI fragments (Labella & Schlessinger 1989) were used for the delineation of rDNA. The EcoRI inserts were cloned into pUC9 and amplified and labeled as described above by PCR (Telenius, 1992). The rDNA probes were directly labeled with diethylaminocoumarin-5-dUTP (DEAC; NEN) and hybridized as described above but without the addition of Cot-1 DNA. Further, a β- satellite probe (86bp) which is located the centromeres of chromosomes 1 , 9, at Yq and all acrocentric chromosomes was labeled chemically using the Biotin Chem Link system according to the manufacturer's instruction. Furthermore, Satellite 1 , 3 and [TTAGGG]π, all-α-satellite-probes were generated and labeled by PCR.

The following stringency conditions have been used:

For low stringency conditions, one hundred nanograms of the labeled probe DNA and 10 ug of salmon sperm DNA in 30% deionized formamide, 2xSSC, and 10% dextran sulfate were denatured by heating to 75°C for 5 min and applied to the slide under a cover glass. Hybridization was carried out at 37°C overnight in a sealed moist chamber. For low-stringency washing conditions the slides were washed three times for 5 min in a 30% , 2xSSC wash solution prewarmed to 45°C and three times for 5 min in 2xSSC at room temperature. Non-specific signal was then blocked by incubation in 4xSSC and 3%BSA for 30 min at 37°C (Protocol 1). It is possible, to denaturate chromosomes for 2-3 min in 70%o formamide, 2xSSC at 80°C, and then to dehydrate in an ethanol series (70%, 90% and 100%). The hybridization solution contained 2ng/μl probe, 50 μg/μl salmon sperm DNA, 50 μg/ml yeast RNA, 30% formamide, 4xSSC, 50mM NaH₂P0₄/Na₂HP0₄, pH 7.0, 1 mM EDTA. Hybridization was performed at 35°C for 14 h, and the slides were washed three times for 5 min in 2xSSC at room temperature (Protocol 2).

For standard stringency conditions, the probe mixture was denatured for 7 min at 75 C and then pre-annealed for about 30 min at 40°C. The slides were denatured in 70%) formamide, 2 x SSC for about 2 min at 70°C. After passage through an ethanol series on ice, the slides were air dried and the hybridization mixture was added to the slide. The hybridization field was sealed with a cover slip and rubber cement, and the slides were incubated at 37°C.

Following hybridization, the slides were washed three times (5 min each) with 4 x SSC Tween at 45°C and then three times (5 min each) with 1 x SSC at 60°C. Blocking was done with 3% BSA in 4 x SSC/Tween for 30 min at 37°C. Afterward, the first layer with rabbit-anti-estradiol (1 :200) in 4 x SSC/Tween plus 1 % BSA was added to the slides. After washing (three times 5 min in 4 x SSC/Tween at 45°C) detection was continued with a second layer consisting of avidin-Cy3.5 (1 :300), anti- rabbit Cy5.5 (1 :400), and anti-digoxigenin Cy7 (1 :100) in 4 x SSC/Tween plus 1 % BSA for 45 min at 37°C. The slides were then washed three times (5 min each) in 4 x SSC/Tween at 45°C, counterstained with DAPI, and embedded in p- phenylenediamine dihydrochloride antifade solution.

Example 5: Reverse M-FISH (ReM-FISH)

Via reverse M-FISH it is possible to analyze the part of the genome which is not covered by M-FISH, viz. the heterochromatin. Traditional methods as e.g. C-Banding or T-Banding, application of a limited number of different repetitive probes with a small number of different fluors are not sufficient to obtain clear results.

Accordingly, the following repeat-depleted probes and/or repetitive probes can, for example, be used: α-satellite, [TTAGGG]_π, rDNA, satellite-pool, pUC1.77, Yq heterochromatin, and 1 p36.

Further a probe can comprise a number of chromosome specific α-satellite probes (which ones may depend on a specific question or sets contain always the same defined probes), [TTAGGG]n, rDNA, satellite-pool, pUC1.77, Yq heterochromatin

(Yqh) and 1p36.

Furthermore, a probe set can consist of number of chromosome specific α-satellite probes, [TTAGGG]_n, rDNA, satellite-pool, pUC1.77, Yq heterochromatin (Yqh) and

1 p36.

Example 6: Complete M-FISH (CoM-FISH)

Complete M-FISH serves to analyze the complete genome within one hybridization. Traditional M-FISH analyzes the euchromatin, and Reverse M-FISH the heterochromatin. Complete M-FISH is designed to collect as many data from the genome in one hybridization as possible. By traditional methods such an analysis is not achievable.

A new probe set could comprise of α-satellite, [TTAGGG]_π, rDNA, satellite-pool, pUCI .77, Yq heterochromatin (Yqh), 1 p36 +rd-M-FISH set (Example set 1). Furthermore, a set might comprise of number of chromosome specific α-satellite probes (which ones may depend on a specific question or sets contain always the same defined probes), [TTAGGG]_n, rDNA, satellite-pool, pUC1.77, Yq heterochromatin (Yqh), 1 p36 +rd-M-FISH set (Example set 2). Another probe set could comprise of number of chromosome specific α-satellite probes, [TTAGGG]_π, rDNA, satellite-pool, pUC1.77, Yq heterochromatin (Yqh), 1 p36 +rd-M-FISH set.

For the M-FISH experiments, pools for each fluorochrome were prepared as described elsewhere (Eils et al., 1998). In brief, the FITC-Pool contained painting probes for chromosomes 1 , 4, 6, 8, 9, 11 , 13, 16, 18, 21 , and Y; the Cy3-Pool, chromosomes 3, 5, 8, 9, 11 , 13, 15, 19, 20, 22, and X; the Bio-Pool, chromosomes 1 , 3, 4, 7, 10, 11 , 15, 17, 19, and Y; the Cy5-Pool, chromosomes 1 , 5, 6, 7, 8, 12, 14, 15, 16, and 22; and the Dig-Pool, chromosomes 2, 3, 5, 6, 9, 10, 12, 21 , X, and Y. Labeling of individual painting probes and the above-mentioned probe pools was carried out by CTA₄-PCR. In biotin- or digoxigenin- (both Boehringer Mannheim) and Cy3- (Amersham Pharmacia Biotech) labeling reactions, 0.08 mM dTTP and 0.02 mM of the respective fluorochrome-dUTPs were used. Cy5 was labeled with 0.068 mM dTTP and 0.032 mM Cy5-dUTP (Amersham). The concentration of dATP, dGTP, and dCTP was 0.1 mM each. FITC labeling was achieved with 0.032 mM FluorX- dCTP (Amersham), 0.068 mM cCTP, and 0.1 mM each of dATP, dGTP, and dTTP.

Appropriate amounts of repetitive probes are added to the repeat-depleted M-FISH (rdM-FISH) mix which only contains repeat-depleted probes to generate a CoM-FISH mix.

CoM-FISH was done as described (Eils et al. 1998; Bolzer et al. 1999) with minor modifications in pretreatment and denaturation of metaphase preparations and posthybridization washes. Pretreatment of slides consisting of a RNase and a pepsin digestion (Lengauer et al. 1992) or pepsin digestion alone appeared to be a crucial determinant for a successful experiment. Depending on the amount of cytoplasm the duration of the pepsin digestion (40 μg/ml) was in the range of 2 to 7 minutes. Probe concentrations in experiments with a single painting probe or five probes was 10-20 ng DNA per probe per microliter of hybridization solution. An empirically determined amount of DNA of each pool was used for M-FISH: FITC, 830 ng; Cy3, 640 ng; Cy5, 830 ng; biotin, 460 ng; and digoxigenin, 730 ng.

A series of FISH experiments was done with various probe concentrations of probe pUC1.77, which is specific for the heterochromatin at band 1q12 on chromosome 1 (Cook and Hindley, 1979).

Five micrograms of salmon testis DNA (Sigma) was added to all probe mixtures. Unlabeled Cot-1 DNA was added only in control FISH experiments of untreated probes hybridized using standard protocols. The probe mixtures were precipitated with ethanol and resuspended in a hybridization solution containing 50% formamide, 2 x SSC, and 15% dextran sulfate. Probe mixtures containing depleted probes or the pUC1.77 probe were denatured but not reannealed and hybridized for one or two nights at 37°C to metaphase chromosome spreads. Hybridization with untreated probes was essentially the same, except that probes were allowed to preanneal for 20 min or longer.

After hybridization, the slides were washed three times with 2 x SSC, 0.1 % SDS at 45°C and then three times with 0.1 x SSC, 0.1 % SDS at 60°C. In experiments with biotin- or digoxigenin-labeled DNA probes, unspecific binding sites were blocked with 3% BSA in 4 x SSC, 0.2%o Tween for 30 min in a moist chamber. Biotin- and digoxigenin-labeled DNA probes were detected with Avidin-Cy3.5 and anti- digoxigenin-Cy7 (both Amersham), respectively. After final washes with 4 x SSC, 0.2%) Tween at 45°C, slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted in phenylenediamine antifade medium.

Example 7: Generation of a repeat-depleted DNA probe

A number of DNA probes can be processed by the method described, including chromosome-specific painting probes generated by microdissection or flow-sorting, any region-specific microdissected probe, cosmids, YACs, PACs, BACs, etc. The CEPH-YAC 933a5 used in this study was provided by Dr. Thomas Haaf from the Max-Planck Institute of Human Genetics in Berlin (http://www.mpimg-beriin- dahlem.mpg-de). The YAC was placed on a pulsed-field gel, after running the human insert was cut from the gel and amplified via DOP-PCR.

In addition, this approach should also work for probes for other species, such as mouse-probes. The DNA is amplified using the method of the present invention. The reaction mixture contained said DNA (usually about 100 ng, however, amount may vary), 5 μl 10x-PCR-buffer (without MgCI₂), 4 μl 25mM MgCI₂, 2 μl 5mM dNTP, 5 μl 6MW-primer 17.0μM (final concentration: 1.7 μM), 0.5 μl Taq Polymerase (2.5 units) and add 50 μl with add H₂0. PCR with 6MW Primer:[5^'-CCG ACT CGA GNN NNN NAT GTG G-3^'] [SEQ ID NO: 1] was then performed by the following conditions: 5 min at 93°C, followed by five cycles of 1 min at 94°C, 1.5 min at 30°C, 3 min transition 30°C-72°C (this refers to the time ramp mentioned above), and 3 min extension at 72°C, followed by 35 cycles of 1 min at 94°C, 1 min at 62°C, and 3 min at 72°C, with an addition of 1 sec/cycle to the extension step and a final extension of 10 min.

However, the conditions of the PCR can be varied as following: The 30°C-72°C transition is elongated to a time longer than 3 min, e.g. 6 min transition time. Cycling conditions are: (1) 94°C for 5 min; (2) five low-stringency cycles of 94°C for 1 min, 30°C for 1.5 min, 6 min transition 30°C-72°C, and 72°C for 3 min; (3) 35 high- stringency cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 3 min, with the addition of 1 s per cycle to the extension time; and (4) a final extension of 5 min at 72°C.

Further, the number of low-stringency cycles can be varied. Following cycling conditions can be performed: (1) 94°C for 5 min; (2) five to ten low-stringency cycles of 94°C for 1 min, 30°C for 1.5 min, 6 min transition 30°C-72°C, and 72°C for 3 min; (3) 35 high-stringency cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 3 min, with the addition of 1 s per cycle to the extension time; and (4) a final extension of 5 min at 72°C. Additionally, the 3 min transition 30°C-72°C can be omitted. Furthermore, the PCR can be done with different primer pairs, e.g. DNA-templates can be amplified with a more stringent version of said PCR called CTA4-PCR (Craig et al. 1997). About 100 ng of the PCR products were amplified by the CTA4-PCR technique according to published protocols (Craig et al. 1997). The conditions were essentially the same as for said PCR. The only differences include the replacement of primer 6MW by the primer 5'-CTA CTA CTA CTA CCG ACT CGA G-3' [SEQ ID NO: 2], annealing was performed at 53°C with or without a time extension, and the number of normal cycles was 36.

Different procedures allow the efficient labeling of DNA-probes. The labeling procedure itself is not important for a successful depletion of repetitive probes. Labeling of individual painting probes and the above-mentioned probe pools was carried out by CTA4-PCR. In biotin- or digoxigenin- (both Boehringer Mannheim) and Cy3- (Amersham Pharmacia Biotech) labeling reactions, 0.08 mM dTTP and 0.02 mM of the respective fluorochrome-dUTPs were used. Cy5 was labeled with 0.068 mM dTTP and 0.032 mM Cy5-dUTP (Amersham Pharmacia Biotech). The concentration of dATP, dGTP, and dCTP was 0.1 mM each. FITC labeling was achieved with 0.032 mM FluorX-dCTP (Amersham Pharmacia Biotech), 0.068 mM cCTP, and 0.1 mM each of dATP, dGTP, and dTTP.

Any variation of aforementioned PCR may be used for the probe labeling. The exact probe and fluor concentrations were described by Eils (1998). Standard nick- translation, random-primed labeling, or Chemical labeling is also suitable. A series of FISH experiments was done with various probe concentrations. Probe concentrations in experiments with a single painting probe or more probes was in the range of 10-20 ng DNA per probe per microliter of hybridization solution. Five micrograms of salmon testis DNA (Sigma) was added to all probe mixtures. Unlabeled Cot-1 DNA was added only in control FISH experiments of untreated probes hybridized using standard protocols. The probe mixtures were precipitated with ethanol and resuspended in a hybridization solution containing 50% formamide, 2xSSC, and 10%-20%> dextran sulfate. Probe mixtures containing depleted probes were denatured but not reannealed and hybridized for one or two nights at 37°C to metaphase chromosome spreads. Hybridization with untreated probes was essentially the same, except that probes were allowed to preanneal for 20 min or longer.

After hybridization, the slides were washed three times with 4xSSC, 0.2% Tween 20 at 45°C and then three times with 1xSSC at 60°C. In experiments with biotin- or digoxigenin-labeled DNA probes, unspecific binding sites were blocked with 3%> BSA in 4xSSC, 0.2%) Tween for 30 min in a moist chamber. Biotin-labeled DNA probes were detected with Avidin-Cy3.5 (Amersham Pharmacia Biotech). Digoxigenin- labeled probes were detected using either one layer of anti-digoxigenin-Cy7 (Amersham Pharmacia Biotech) or with a different, two layer system consisting of anti-dig rabbit (Sigma) in the first step and anti-rabbit Cy5.5 (Amersham Pharmacia Biotech) in a second step. After final washes with 4xSSC, 0.2% Tween at 45°C, slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and mounted in phenylenediamine antifade medium. The fluors used are not decisive for the outcome of the experiment, it should work with any fluor from any manufacturer. The images of the hybridized metaphase spreads were captured using a Leica DMRXA-RF8 microscope equipped with a cooled Sensys CCD camera (Photometries) controlled by Leica QFISH software (Leica Microsystems Imaging Solutions Ltd.), as described elsewhere (Eils et al., 1998).

Further, the images were analyzed using the Leica image analysis software programs QFISH, QWIN, and MCK (Leica Microsystems Imaging Solutions Ltd.). References

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Claims

Claims

1. A method for the visualization of eu- and heterochromatin comprising hybridizing to a target DNA a repeat-depleted probe and a probe specific for repetitive sequences.

2. The method of claim 1 , wherein the hybridizing of the a repeat-depleted probe and the probe specific for repetitive sequences occurs under the same hybridizing conditions.

3. The method of claim 1 or 2, wherein the a repeat-depleted probe and the probe specific for repetitive sequences are hybridized simultaneously.

4. The method of any one of claims 1 to 3, wherein the hybridizing is an in situ hybridization.

5. The method of claim 4, wherein the in situ hybridization is a fluorescence in situ hybridization (FISH).

6. The method of any one of claims 1 to 5, wherein the repeat-depleted probe is a chromosome painting probe.

7. The method of any one of claims 1 to 6, wherein the probe is a combination of probes.

8. The method of any one of claims 1 to 7, wherein the target DNA comprises DNA of one or more individual chromosomes.

9. The method of claim 8, wherein the target DNA comprises a complete chromosomal set.

10. The method of any one of claims 1 to 8, wherein the repeat-depleted probe is specific for one or more acrocentric chromosomes or for one or more chromosomes or chromosomal regions.

11. The method of any one of claims 1 to 10, wherein the target DNA is derived from animals or plants.

12. The method of any one of claims 1 to 11 , wherein the target DNA is derived from tumors, oocytes, sperms, embryonic tissues or cells obtained by amniocentesis.

13. The method of any one of claims 1 to 12, wherein the target DNA is stained.

14. Use of the method of any one of claims 1 to 13 for the simultaneous visualization of eu- and heterochromatin, for the painting of individual chromosomes, in situ hybridization, FISH, M-FISH, CoM-FISH, ReM-FISH, tumor cytogenetics, or pre- or postnatal diagnostic.

15. Use of a combination of repeat-depleted probe and a probe specific for repetitive sequences for the visualization of eu- and heterochromatin.

16. Use of a repeat-depleted probe and a probe specific for repetitive sequences for the preparation of a composition for the identification of polymorphism, structural or complex chromosomal rearrangement, of supernumerary marker chromosomes (SMC), for pre- or postnatal diagnostic, for visualization of the p-arms of arcocentric chromosomes, or tumor cytogenetics.

17. A composition comprising a repeat-depleted probe and a probe specific for repetitive sequences.

18. The use of claim 16 or the composition of claim 17, wherein the composition is a diagnostic composition.

19. A kit comprising an instruction for performing the method of any one of claims 1 to 13.

20. A kit comprising a repeat-depleted probe and a probe specific for repetitive sequences.

21 Use of the kit of claim 19 or 20 for the performing of the method of any one of claims 1 to 13.