Comparative genomic hybridization (CGH) is a molecular cytogenetic method used to analyze copy number variations (CNVs) across the entire genome. CNVs, which include deletions and duplications of DNA segments, are implicated in various diseases, including cancer and developmental disorders.
Principles of CGH
CGH involves the competitive hybridization of differentially labeled DNA from a test sample and a reference sample to normal metaphase chromosomes. The test DNA is labeled with one fluorescent dye (e.g., green), while the reference DNA is labeled with a different dye (e.g., red). Both labeled DNAs are then mixed and co-hybridized to the metaphase chromosomes. Repetitive DNA sequences are blocked using Cot-1 DNA to ensure specific hybridization.
The fluorescence intensities of the two dyes are measured along each chromosome. A balanced ratio of green and red fluorescence indicates equal copy numbers in the test and reference samples. An increased green-to-red ratio suggests a gain of DNA in the test sample, whereas an increased red-to-green ratio indicates a loss of DNA in the test sample. This ratio is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes.
Applications of CGH
CGH has been widely used in the study of human and mouse cancers to identify genomic imbalances that may contribute to tumor development and progression. These imbalances can involve oncogenes (genes that promote cancer) and tumor suppressor genes (genes that inhibit cancer). CGH can also detect CNVs associated with developmental disorders like Down syndrome, Prader-Willi syndrome, Angelman syndrome, and Cri-du-Chat syndrome.
Limitations of Traditional CGH and the Development of Array CGH
Traditional CGH using metaphase chromosomes has limitations in resolution. It can only detect relatively large CNVs (greater than 20 Mb). Smaller aberrations and closely spaced changes are difficult to discern. Linking ratio changes to specific genomic markers is also challenging. To overcome these limitations, array CGH was developed.
Array CGH utilizes an array of mapped DNA sequences instead of metaphase chromosomes. This allows for higher resolution analysis of CNVs, enabling the detection of much smaller genomic alterations. The resolution in array CGH is determined by the density of the probes on the array. The test and reference DNA are hybridized to the array, and the fluorescence ratios are measured for each probe. This provides a detailed map of copy number changes across the genome.
Conclusion
Comparative genomic hybridization is a powerful technique for analyzing DNA copy number variations throughout the genome. While traditional CGH using metaphase chromosomes has limitations in resolution, array CGH offers a higher resolution approach, allowing for the detection of smaller genomic imbalances and providing more precise mapping of CNVs. This technology has significantly advanced our understanding of the role of CNVs in various diseases, particularly in cancer and developmental disorders.
