Comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique used to screen the entire genome for gains and losses of genetic material (1). It is being used increasingly in the study of cancer genetics to identify genes important in the initiation, progression, and, of particular relevance here, metastasis of tumors (2-4).One of the advantages of the technique is that the entire genome is examined in a single experiment, so there is no necessity to know the genetic region of interest prior to investigation. Once regions of gain or loss have been identified, these regions can be defined further using fluorescence in situ hybridization (FISH) (described in Chapter 14 by Goker and Shipley) or molecular genetic techniques. CGH is essentially a modified in situ hybridization. Differentially labeled test or tumor DNA (green) and reference or normal DNA (red) are cohybridized to normal metaphase spreads. Differences in the copy number between test and reference DNA are seen as differences in the ratio of green to red fluorescence intensity on the metaphase chromosomes. Images of the metaphases are captured and quantification of the fluorescence ratios performed using a digital image analysis system. Regions of chromosomal gain are seen as an increased fluorescence ratio whereas regions of loss are seen as a decrease in the fluorescence ratio. Losses are detectable when the region affected exceeds 10 Mb (5), smaller regions of gain are detected if there is high level amplification, for example a 2-Mb region that is amplified five times will be visualized (6). For each tumor, 5/210 metaphases are analyzed and an average fluorescence ratio for each chromosome obtained.