FISH uses DNA probes that recognize sequences specific to chromosomal regions of greater than 100 kilobases in size, which defines the limit of resolution with this technique for identifying chromosomal changes. Such probes are labeled with fluorescent dyes and are applied to meta-phase spreads or interphase nuclei. The probe hybridizes to its complementary sequence on the chromosome and thus labels the specific chromosomal region that can then be visualized under a fluorescence microscope. The ability of FISH to circumvent the need for dividing cells is invaluable when a rapid diagnosis is warranted (e.g., in a critically ill infant suspected of having an underlying genetic disorder). Such analysis can be performed on prenatal samples (e.g., cells obtained by amniocentesis, chorionic villus biopsy, or umbilical cord blood), peripheral blood lymphocytes, and even archival tissue sections. FISH is used for the detection of numeric abnormalities of chromosomes (aneuploidy) (
Fig. 7.38A ), subtle microdeletions (
Fig. 7.38B ) and complex translocations not indentifiable by routine karyotyping, and gene amplification (e.g., MYCN amplification in neuroblastomas).
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FISH requires previous knowledge of the one or few specific chromosomal regions suspected of being altered in the test sample. However, genomic abnormalities can also be detected without previous knowledge by using micro-array technology to perform a global genomic survey. First-generation platforms were designed for comparative genomic hybridization (CGH), whereas newer platforms incorporate single nucleotide polymorphism (SNP) genotyping approaches, offering multiple benefits.
In array comparative genomic hybridization (aCGH), the test DNA and a reference (normal) DNA are labeled with two different fluorescent dyes that fluoresce red and green, respectively. The differentially labeled samples are then cohybridized to an array spotted with DNA probes that span the human genome at regularly spaced intervals, and usually cover all 22 autosomes and the sex chromosomes. At each chromosomal probe location, the binding of the labeled DNA from the two samples is compared. If the two samples are equal (i.e., the test sample is diploid), then all spots on the array will fluoresce yellow (the result of an equal admixture of green and red dyes). In contrast, if there is even a focal deletion or duplication, the probe spots corresponding to it will show skewing toward red or green (depending on gain or loss of material), allowing highly accurate determinations of copy number variants across the genome.
Newer types of “SNP chips” are based on a similar concept, but the probes are designed to identify SNP sites genomewide, which has a number of advantages over CGH. As discussed in Chapter 1, SNPs are the most common type of DNA polymorphism, occurring approximately every 1000 nucleotides throughout the genome (e.g., in exons, introns, and regulatory sequences). Thus, SNPs serve both as a physical landmark within the genome as well as a genetic marker whose transmission can be followed from parent to child. There are several testing platforms that allow SNPs to be analyzed genomewide on arrays. Like CGH probes, methods involving SNPs can be used to determine copy number variations. In addition, by discriminating between SNP alleles at each particular location, they also provide zygosity data (
Fig. 7.39 ). This means, for example, that SNP chips can identify uniparental disomy, in which loss of heterozygosity is seen within an affected region having diploid DNA content. The current generation of SNP arrays is quite comprehensive, with the