Chapter 063. Chromosome Disorders (Part 2) Molecular Cytogenetics The introduction of FISH methodologies in the late 1980s revolutionized the field of cytogenetics. In principle, FISH is similar to other DNA-DNA hybridization methodologies. The probe is labeled with a hapten, such as biotin or digoxigenin, to allow detection with a fluorophore (e.g., FITC or rhodamine). After the hybridization step, the specimen is counter-stained and the preparations are visualized with a fluorescence microscope. Types of FISH Probes A variety of probes are available for use with FISH, including chromosome-specific paints (chromosome libraries), repetitive probes, and single- copy probes (Fig. 63-2). Chromosome libraries hybridize to sequences that span the entirety of the chromosome from which they are derived and, as a result, they can be used to "paint" individual chromosomes. Figure 63-2 Examples of different applications of fluorescence in situ hybridization (FISH) to human metaphase and interphase preparations. A, B. Aneuploidy detection: Interphase FISH using chromosome 13 (green) and chromosome 21 (red) unique sequence probes on interphase cells from direct amniotic fluid preparations. In "A" (a normal cell), two signals for both chromosomes 13 and 21 are seen; in "B," three signals for chromosome 21 are seen, indicating trisomy 21 in the fetus. C. Aneuploidy detection: Two-color FISH with telomere probes from the short arm (green) and the long arm (red) of chromosome 8. Hybridization with these probes shows fluorescence of both probes to three separate chromosomes, indicating the presence of trisomy 8 in this individual. D. Microdeletion detection: Two-color FISH is used to detect a microdeletion of chromosome 22 associated with velocardiofacial (VCF) syndrome. A probe for ARSA (a locus on the distal portion of chromosome 22, visualized as a green signal) is observed on both chromosomes 22. However, a probe for TUPLE1 (a locus within the VCF region of chromosome 22, visualized in red) hybridizes to only the normal chromosome. E. Characterization of structural rearrangements: M-FISH (multicolor FISH) is used to detect a complex chromosome rearrangement involving a translocation between chromosome 6 and 16, as well as a translocation and inversion involving chromosomes 2 and 10. Repetitive probes recognize amplified DNA sequences present in chromosomes. The most common are α-satellite DNA probes that are complementary to DNA sequences found at the centromeric regions of all human chromosomes. A vast number of single-copy probes are now available as a result of the human genome project. These probes can be as small as 1 kb, though normally they are much larger and are packaged into cosmids (40 kb), bacterial artificial chromosomes (BACs) or P1 clones (100–200 kb), or yeast artificial chromosomes (YACs) (1–2 Mb). Many are available commercially, including probes for a variety of microdeletion syndromes and for subtelomeric regions of individual chromosomes. . Chapter 063. Chromosome Disorders (Part 2) Molecular Cytogenetics The introduction of FISH methodologies in the. including chromosome- specific paints (chromosome libraries), repetitive probes, and single- copy probes (Fig. 63 -2). Chromosome libraries hybridize to sequences that span the entirety of the chromosome. using chromosome 13 (green) and chromosome 21 (red) unique sequence probes on interphase cells from direct amniotic fluid preparations. In "A" (a normal cell), two signals for both chromosomes