Fluorescence In Situ Hybridization

Abstract

The 21st century is witnessing emergence of a new era in medicine called “Genomic Disease Management.” Chromosomes, which house the essential components of this new faculty of medicine, have gained scientific attention since the late 19th century. However, it took nearly 100 yr before the understanding of these vital molecules could be taken to a patient’s bedside. The association of chromosomal abnormalities with congenital malformation disorders and cancer has been suspected since the late 1950s (1–4). In particular, demonstration by these studies of trisomy of one of the smallest chromosomes in Down’s syndrome (chromosome 21, as shown later), monosomy X in Turner’s syndrome, and the chromosomal analysis of leukemias laid foundation for the coming years. It was the development of a variety of staining methods assigning uniqueness to individual chromosomes that really marked the beginning of genomic analysis (for review, see ref. 5). As shown in Table 1, these techniques are collectively known as chromosome banding. A realization that using certain stains like Giemsa and Quinacrine mustards, the AT-rich and GC-rich regions of chromosomes could be distinguished into a unique banding pattern for every chromosome, indeed, invited clinical interest into this fascinating field (6–7). Using a metaphase cell preparation, it became possible to clearly identify chromosomal abnormalities in clinical specimens. The banding techniques underscored the fact that the association of chromosomal abnormalities with disease might not be random or an epiphenomenon. A demonstration of two consistent chromosomal translocations—t(9;22) related to Philadelphia chromosome in chronic myeloid leukemia (CML) and t(15;17) found in acute promyelocytic leukemia (APL)—provided direct testimony for a specificity of chromosomal anomalies in hematological malignancies (4,8,9). Ever since, chromosomal banding became the gold standard for both clinical and basic science studies in cytogenetics. Many authors view the progress of cytogenetics in three phases: (1) the Prebanding era (until 1970), (2) the banding era (1970–1980), and (3) the present-day constantly evolving molecular cytogenetics era (post-1980) (10,11). The roots of this astoundingly progressive molecular phase in the last two decades actually could be seen as early as 1969, when the concept of in situ oligonucleotide hybridization (ISH) was introduced by several groups simultaneously (12–14). Albeit, the use of radioisotopic labeling and the autoradiographic visualization method of ISH restricted its general application. Between 1986 and 1988, two major advances illuminated the field of cytogenetics: (1) the development of nonradioactive fluorescent oligonucleotide probe labeling technique and (2) the construction of human-chromosomespecific libraries (15–18). During this period, the concept of “interphase cytogenetics” was born, which offered a tremendous technical ease in the ISH protocol and reduced the assay time considerably (17). The fluorescence in situ hybridization (FISH) has since come a long way, metamorphosing from a concurrent detection of several chromosomal abnormalities to the identification of previously uncharacterized abnormalities with multiplex-FISH (M-FISH) or spectral karyotyping (SKY™), further to genomic screening with FISH-based metaphase-comparative genomic hybridization and most recently to the solid-phase genomic DNA arrays (for review, see refs. 11 and 19). The present chapter describes the principles of ISH and FISH and their potential clinical applications and, finally, introduces the seeds of future developments in FISH technology.