Combinatorial labeling principle
The left side of the figure depicts the principle behind combinatorial labeling. Using either three haptene-labeled nucleotides (BIO-dUTP, DIG-dUTP or DNP-dUTP) or three fluorescent-labeled nucleotides (FITC-dUTP, Cy3-dUTP and AMCA-dUTP), seven different DNA probes can be labeled and simultaneously identified after hybridization, based on color combinations. For example, a DNA probe labeled with FITC will fluoresce green, another one labeled with AMCA will fluoresce blue, whereas a third one labeled with FITC and AMCA will fluroesce cyan. Similarly, the combination betwen red and green yields yellow, the one between blue and red yields magenta, whereas a probe labeled with all three fluors (FITC-green, AMCA-blue and Cy3-orange/red) will fluoresce "white". In combinatorial labeling, the number of all possible combinations is described by the formula shown in the figure. In FISH, however, a DNA probe is usually labeled with no more than three fluorescent dyes at the same time, so the number of usable combinations is smaller. Note: the haptens do not fluoresce themselves; DNA labeled with them will be detected by using antibodies labeled with the appropriate fluors (which can be the same, FITC, Cy3 and AMCA, or many other ones).
The right side of the figure shows two hybridizations, which demonstrate the principle and usefulness of combinatorial labeling. (1) In the upper image, three centromeric probes were labeled, each with one fluor (14/22 probe with FITC/green, X probe with Cy3/red and Yprobe with AMCA/blue) and were simultaneously hybridized on a human metaphase. The probes are easily distinguished (Note: the 14/22 alpha-satellite probe hybridizes to the centromeres of chromosome pairs 14 and 22, yielding four hybridization signals. By comparison, a male metaphase contains only one chromosome X and one chromosome Y = which are the two sex chromosomes). (2) In the bottom image, DNA probes for the centromeres of chromosomes 15 (FITC/green), 17 (Cy3/red) and 18 (FITC and Cy3) were simultaneously hybridized onto human cells. The image depicts a nucleus (arrow), showing two green (chromosome 15), two red (chromosome 17) and two yellow (chromosome 18) fluorescent dots. Because the DNA probe for the centromere of chromosome 18 was labeled with both dyes (combinatorial labeling), the DNA probe for this choromosome emits both green and red (green + red = yellow), thus chromosome 18 centromere can be differentiated from the other two chromosomes although only two flors (FITC and Cy3) were used to label the three DNA probes .