Chromosome painting

Chromosome painting involves the use of fluorescent-tagged chromosome specific DNA sequences to visualize specific chromosomes or chromosome segments by in situ DNA hybridization and fluorescence microscopy. Chromosome painting refers to the hybridization of fluorescently labelled chromosome-specific, composite probes to cytological preparations.

Chromosome painting allows the visualization of individual chromosomes in metaphase or interphase stages and the identification of both numerical and structural chromosomal aberrations with high sensitivity and specificity. The simultaneous hybridization of multiple chromosome painting probes, each tagged with a specific light-emitting fluorochrome has resulted in the differential colour display of human and mouse chromosomes, which is also called colour karyotyping.

Fluorescent in situ hybridization (FISH) has been used to detect the location of specific genomic targets using probes that are labelled with specific fluorochromes. That is the reason chromosome painting is also called M-FISH or multicolour FISH. The technique allowed detection of simple and complex chromosomal rearrangements. In addition, complex chromosomal abnormalities could also be identified that could not be detected by the conventional cytogenetic banding techniques.

Almost a decade ago, chromosome painting was developed independently by research teams at Lawrence Livermore National Laboratories and at Yale University. Both groups had taken advantage of the availability of cloned DNA libraries that were derived from flow-sorted human chromosomes. The first generation of probes, based on chromosome-specific phage libraries, were rather cumbersome to use, due to low insert-to-vector ratios, which frequently resulted in a relatively high background staining. Some of these limitations were overcome with the availability of plasmid libraries, where an improved insert-to-vector ratio and easier probe generation enhanced the painting quality considerably.

Chromosome painting has improved the efficiency of screening cells for chromosome abnormalities, in testing chemicals for mutagenacity and for rearrangements associated with tumours. Painting probes detect chromosome rearrangements. Use of the same chromosome paints for chromosomes of different species reveals the extent of chromosome rearrangements since divergence of the species.

Chromosome painting probes are now also available for an ever increasing number of species, most notably for the mouse and the rat, allowing the expansion of chromosome painting analyses to animal models for human diseases. FISH techniques have been developed and applied to identify the origin of the markers and other structural chromosomal aberrations. The use of chromosome painting probes in one, two or three colour FISH experiments has significantly improved the definitive diagnosis of chromosomal aberrations.

The introduction of chromosome painting to the field of comparative cytogenetics has added significantly to the understanding of chromosome changes that occurred during the evolution of species. Chromosome painting can be used to identify homologous chromosome segments in different species and to map probes of different complexities and chromosome rearrangements in a single experiment.  In recent years, the complete karyotypes of various mammals including primates, carnivores and artiodactyls have been analyzed by chromosome painting.

Chromosome Painting Probes, available in liquid format, are directly labelled in either a red or green fluorophore. They can be mixed together to label a number of chromosomes in a single reaction. The probes come in the form of ready to use hybridization solution in a five-test kit format, and are supplied complete with DAPI counter stain. The protocol is rapid and simple and follows simultaneous co-denaturation of the FISH probe and target DNA.

The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes micro-dissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). The chromosomal material is amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products are labelled by nick-translation with biotin-11-dUTP and used as probes for FISH.

Homologies between the chromosomes of different species can be detected by chromosome painting. A more detailed description of how this method of chromosome painting works is given below:

· Initially suspensions of chromosomes from dividing cells are sorted by a method known as flow cytometry.

· DNA from one chromosome is then labelled with a fluorescent dye using a technique called fluorescence in situ hybridisation (FISH). This labelled DNA paints the chromosome and allows homologous regions of DNA of other species, from great apes to mice, to be identified. Homologous regions show up in the same colour on the chromosome charts.

· If you take a paint made from human chromosome 2 and hybridise it onto the chromosomes of another species, then you’ll see segments of paint on different chromosomes, each being homologous to part of human chromosome 2, so with the gibbon karyotype, regions on chromosome 2 in human can be tracked to 5 different chromosomes in gibbon. The fluorescent-labelled DNAs will attach to the analogous chromosomes from which they were derived. DNA fragments with the same base sequences have the characteristic of attaching to each other.

· This tells us that the lineage that produced gibbon and the lineage that produced human have diverged over several million years and during this time rearrangements have occurred between the two species which can be tracked using the painting technique.

· If a part of a painted chromosome (yellow, for example) had undergone an exchange with another, non-painted chromosomes (stained red), it is possible to detect the aberration as reciprocal translocation, because the aberrant chromosome contains both yellow and red segments. Usually, a pair of bi-coloured chromosomes can be detected in one metaphase, because two chromosomes typically exchange a part of their DNA. Reciprocal translocations are difficult to detect by simple staining technique that stains the entire set of chromosomes with a single material such as with Giemsa.

· When human chromosome probes are hybridised to the chromosomes from other species, the same set of blocks, dispersed across multiple human chromosomes, are often located together on one chromosome in other species. For example, parts of human chromosome 3 and 21, or 14 and 15, or 12 and 22, tend to be located together on one chromosome in other animals. This is evidence that an ancestral block of genes has been split apart and moved to different chromosomes during human evolution.

· Collating all these patterns of linked regions of chromosome across various mammalian species has enabled to assemble a picture of genomic commonalities across multiple species.

Chromosome Walking

A technique that helps to identify overlapping cloned DNA fragments from one continuous segment of a chromosome. These fragments are generated by random shearing or by partial digestion with a restriction endonuclease. A series of colony hybridisations is carried out, starting with a specific cloned fragment that has already been identified and which is known to be in the region encompassed by the overlapping clones. This identified fragment is used as probe to identify clones containing adjacent sequences. These are in turn used as probes to identify other clones carrying sequences adjacent to them and so on. With each round of hybridisation, it is possible to “walk” further along the chromosome from the initial fragment. This technique was developed in the late 1980s and is a powerful method to detect translocations.