Karyotyping from peripheral blood is a standard technique used to examine human chromosomes and to identify chromosomal abnormalities (1). A small amount of anticoagulated blood is centrifuged to isolate white blood cells, to which are added tissue culture medium containing plant lectins such as phytohemagglutinin to stimulate them to divide. The dividing cells are then arrested at metaphase with mitotic spindle inhibitors such as colchicine and are lysed in hypotonic buffers to prepare metaphase chromosomes. Preparations of metaphase chromosomes are then stained with dyes such as Giemsa (G-banding) and are examined under a microscope. G-banding involves a series of acid, heat, or gentle trypsin treatment of chromosome preparations followed by staining with Giemsa. The alternation of chromosomal regions with high or low G/C content results in a pattern of light and dark bands that is unique for each human chromosome (Fig. 94.2A). The unique pattern for each human chromosome allows the unequivocal definition of each chromosome, and any deviations from this typical pattern may indicate occurrence of chromosomal abnormalities (Fig. 94.2B). Other chromosome banding techniques producing quinacrine (Q) bands, reverse (R), or centromeric (C) bands can be used to examine special chromosomal domains.

Fluorescence in situ hybridization (FISH) technology is a major tool for gene mapping and for identifying chromosomal abnormalities (2,3,4). In FISH analysis, DNA probes (any cloned DNA sequences, genes, microdissected fragments, or flow-sorted chromosomes) are labeled by incorporation of chemically modified nucleotides (e.g., 5-bromo-2-deoxyuridine) that fluoresce directly or can be labeled by incorporation of reporter molecules (e.g., digoxigenin or biotin). The DNA probes are then hybridized to metaphase or interphase chromosomes. Chromosomes are then counterstained with 4,6-diamino-2-phenylindole to provide high-definition banding, and hybridization signals are detected by binding a fluorescently tagged molecule to reporter molecules (e.g., anti-digoxigenin-fluorescein isothiocyanate). Chromosomes are then photographed under a fluorescence microscope (Fig. 94.2C). FISH can allow rapid mapping of any gene or DNA sequence cloned into cosmids, bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs) to a particular chromosome (Fig. 94.2C). FISH can also be used to detect numeric chromosomal abnormalities, including the small deletions involved in DiGeorge syndrome and Williams syndrome.

Advances in FISH technology include chromosome painting, which allows the entire chromosome to fluoresce with the chosen color (Fig. 94.2C), and multicolor FISH, which allows each human chromosome to be painted in up to 12 different colors (2). Both chromosome painting FISH and multicolor FISH can be used to identify complex chromosomal rearrangements or abnormalities rapidly. In conclusion, both karyotype analysis and FISH are powerful technologies that can be implemented in many research and diagnostic fields of human genetics.

Until the mid-1980s, the primary approach to identify the gene responsible for a human disease was through careful analysis of biochemical and physiologic differences between healthy and affected individuals. The deficient protein or enzyme from patients can be purified, and partial amino acid sequences can be obtained or antibodies against the protein can be raised. The antibodies, degenerate oligonucleotides derived from the partial amino acid sequences, or tagged fusion proteins, can then be used to screen various complementary DNA (cDNA) libraries to identify the disease gene. The approach moving from the basic biochemical defect to the disease gene is referred to as functional cloning. Sickle cell anemia caused by mutations in the hemoglobin gene was the first human disease characterized by human molecular genetics. Initially, red blood cells from affected individuals were noticed to be abnormally shaped. Later, it was found that hemoglobin from patients was electrophoretically different from normal hemoglobin, and this difference was the result of the substitution of valine for glutamic acid at the sixth amino acid position of the β-chain of hemoglobin.
Subsequently, the gene for β-hemoglobin was cloned, and the mutation of a single base substitution of A to T was identified at the nucleic acid level (5). However, the genetic origin of most diseases cannot be determined by functional cloning because of the lack of information on biochemical defects.