Chapter 063. Chromosome Disorders (Part 2)

Molecular CytogeneticsThe 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 ProbesA variety of probes are available for use with FISH, including chromosome-specific paints (chromosome libraries), repetitive probes, and singlecopy probes (Fig. 63-2). Chromosome libraries hybridize to sequences that span the entirety of the...
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Chapter 063. Chromosome Disorders (Part 2) Chapter 063. Chromosome Disorders (Part 2) Molecular Cytogenetics The introduction of FISH methodologies in the late 1980s revolutionizedthe field of cytogenetics. In principle, FISH is similar to other DNA-DNAhybridization methodologies. The probe is labeled with a hapten, such as biotin ordigoxigenin, to allow detection with a fluorophore (e.g., FITC or rhodamine).After the hybridization step, the specimen is counter-stained and the preparationsare visualized with a fluorescence microscope. Types of FISH Probes A variety of probes are available for use with FISH, includingchromosome-specific paints (chromosome libraries), repetitive probes, and single-copy probes (Fig. 63-2). Chromosome libraries hybridize to sequences that spanthe entirety of the chromosome from which they are derived and, as a result, theycan 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. Aneuploidydetection: Interphase FISH using chromosome 13 (green) and chromosome 21(red) unique sequence probes on interphase cells from direct amniotic fluidpreparations. In A (a normal cell), two signals for both chromosomes 13 and 21are seen; in B, three signals for chromosome 21 are seen, indicating trisomy 21in the fetus. C. Aneuploidy detection: Two-color FISH with telomere probes fromthe short arm (green) and the long arm (red) of chromosome 8. Hybridization withthese 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 associatedwith velocardiofacial (VCF) syndrome. A probe for ARSA (a locus on the distalportion of chromosome 22, visualized as a green signal) is observed on bothchromosomes 22. However, a probe for TUPLE1 (a locus within the VCF regionof chromosome 22, visualized in red) hybridizes to only the normal chromosome.E. Characterization of structural rearrangements: M-FISH (multicolor FISH) isused to detect a complex chromosome rearrangement involving a translocationbetween chromosome 6 and 16, as well as a translocation and inversion involvingchromosomes 2 and 10. Repetitive probes recognize amplified DNA sequences present inchromosomes. The most common are α-satellite DNA probes that arecomplementary to DNA sequences found at the centromeric regions of all humanchromosomes. A vast number of single-copy probes are now available as a resultof the human genome project. These probes can be as small as 1 kb, thoughnormally they are much larger and are packaged into cosmids (40 kb), bacterialartificial chromosomes (BACs) or P1 clones (100–200 kb), or yeast artificialchromosomes (YACs) (1–2 Mb). Many are available commercially, includingprobes for a variety of microdeletion syndromes and for subtelomeric regions ofindividual chromosomes.