Molecular cytogenetics
Molecular cytogenetics involves the combination of molecular biology and cytogenetics. In general this involves the use of a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which we use DNA probes labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. FISH can either performed as a direct approach, in which we use either metaphase chromosomes or interphase nuclei, or as an indirect approach, which involves the use of comparative genomics hybridization (CGH) analysis to asses the entire genome for of imbalanced chromosomal material.
Metaphase FISH
Metaphase and interphase FISH are "direct" approaches that make use of chromosome-specific paints or single locus probes. These are powerful techniques that allow a rapid assessment of the gross numerical and structural characteristics of a cell, looking directly at the chromosomes of the tumor cells Tumor metaphase FISH requires the availability of viable cells from which to produce tumor chromosome preparations for analysis. Interphase FISH is able to make use of preserved cellular material and may thus be extended to include the use of paraffin embedded biopsy sections. In the examples below, the figure on the left shows the application of FISH of single locus probes (SLP) to determine the number of copies of three different chromosomes in a canine cancer metaphase spread. In this case the chromosome with a blue SLP is present as three copies and so the cell is trisomy for this chromosome. On the right hand side is an example of a five color FISH reaction, using chromosome paint probes, to identify the nature of multiple aberrant chromosome in a metaphase preparation of a canine tumor cell.
Interphase FISH
As with metaphase FISH, interphase FISH is a ‘direct’ approach where single locus probes (SLPs) are use to probe cell nuclei to assess the gross numerical and structural characteristics of a tumor cell population. In contrast to metaphase FISH interphase FISH is able to make use of preserved cellular material and may thus be extended to include the use of archival paraffin embedded biopsy sections. The example below shows the application of multicolor FISH of single locus probes (SLP) to determine the number of copies of five different chromosomes in a canine cancer cell nucleus. In this case the chromosomes represented by red, orange and pink SLPs all have a normal copy number of n=2, while the chromosomes represented by a blue and a green SLP are both present as three copies, indicating that both these chromosome are trisomy in this cell.
