Comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a screening method based on fluorescence in situ hybridization (FISH). In contrast to conventional FISH, the metaphase target is derived from a normal peripheral blood lymphocyte culture. This target is hybridized to the test or tumor DNA, which is labeled/detected by one fluorochrome (i.e., green), and to an equal amount of labeled normal or reference DNA, which is labeled/detected by a different fluorochrome (red). It is the difference in these green/red ratios (determined by specialized software) along the length of each karyotyped chromosome that indicates the relative copy number changes in the test/tumor DNA. The basic FISH techniques reviewed in this section, the parameters for which also apply to obtaining satisfactory results for CGH, include cytogenetic preparation and slide-making, DNA extraction (from fresh or paraffin-embedded tissues) and labeling, slide pretreatment, hybridization, post-hybridization washes, and detection.

Current status of fluorescent in-situ hybridisation.

Several modifications have taken place in fluorescent in-situ hybridisation (FISH) techniques, including comparative genomic hybridisation, primed in-situ labelling, interphase FISH, multicolour FISH and in combination with peptide nucleic acid technology. FISH can also be combined with microarrays. Selected innovative technologies are described. The most clinically important applications are in cytogenetics and oncology.

Multicolor chromosome painting in diagnostic and research applications.

For many years whole chromosome painting probes have been the work-horses in a large variety of clinical and research molecular cytogenetic applications. In recent years painting probes have been complemented by an increasing number of further region-specific probes, which allow the specific staining of centromeres, subtelomeres or other regions within the genome. This development of new probe sets was greatly facilitated by the Human Genome Project from which well-characterized probes for any region within the genome have emerged. Furthermore, the evolution of different multicolor fluorescence in situ hybridization (FISH) technologies now allows the cohybridization of multiple DNA-probes of different colors. These developments have paved the way for FISH-based automated karyotyping or the simultaneous analysis of multiple defined regions within the genome. Using appropriate instrumentation and image processing, the analysis can be performed two-dimensionally on metaphase spreads or three-dimensionally in intact interphase nuclei. Here we summarize some of the most recent developments and discuss the application of painting probes in different scenarios.

Refined characterisation of chromosome aberrations in tumours by multicolour banding and electronic mapping resources.

Acquired chromosome abnormalities in tumours often reflect pathogenetic events at the gene level. Multicolour fluorescence in situ hybridisation (FISH) with single-copy probes offers extensive possibilities to characterise chromosome breakpoints in relation to the physical map of the human genome. This approach is based on the construction of comprehensive EST- based maps, combinatorial labelling of probes, and tumour cell preparations optimised for metaphase FISH. Information from several electronically available databases is combined into an integrated physical map, to which clones carrying yeast and bacterial artificial chromosomes are anchored. Extracted DNA or PCR products from these clones are then fluorescently labelled by one or several fluors, allowing simultaneous FISH detection of multiple loci. To improve hybridisation efficiency and reduce background fluorescence, standard methods for chromosome preparation from cultured tumour cells are complemented with a prolonged trypsin treatment to obtain complete disaggregation of cells, and exposure of the metaphase spreads to detergent and saline at high temperature, followed by pepsin digestion to remove extracellular matrix and cytoplasmic debris. The resulting colour-banding allows the characterisation of chromosome abnormalities in relation to expressed sequences, even in tumours exhibiting highly complex rearrangements.

Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing.

BACKGROUND: Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. RESULTS: This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. CONCLUSION: The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.

IPM-FISH, a new M-FISH approach using IRS-PCR painting probes: application to the analysis of seven human prostate cell lines.

We have developed an alternative multicolor karyotyping technique based on multiplex fluorescence in situ hybridization (M-FISH) and our own optical device with a specific filter set. The most innovative part of our development is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous to the combinatorial labeling. This allows us not only to recognize the origin of chromosomal fragments, but to identify the breakpoints as well. We have used this technique to analyze seven cell lines: four prostate cancer cell lines (CA-HPV-10, LNCaP, DU145, and PC3), and three normal transformed epithelial prostate cell lines (PNT1B, PNT2, and PZ-HPV-7). In order to validate our IRS-PCR multiplex FISH (IPM-FISH) technique and to complement the results, we applied comparative genomic hybridization (CGH) and FISH analysis, showing good correlation with the IPM-FISH results. To date, molecular and cytogenetic studies have identified several chromosomal regions that are altered in human prostate cancer; several candidate genes have been suggested. However, reliable markers for predicting the aggressiveness of early prostate cancer are not yet available. Our results show several common, unbalanced rearrangements in the cell lines. These rearrangements are similar to regions already implicated in prostate cancer, validating these cell lines as a good model system.

ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties.

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.