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  • Title: Analysis of genomic aberrations using comparative genomic hybridization of metaphase chromosomes.
    Author: Carless MA.
    Journal: Methods Mol Biol; 2015; 1288():67-94. PubMed ID: 25827876.
    Abstract:
    Comparative genomic hybridization (CGH) allows the global screening of copy number aberrations within a sample. Specifically, large (>20 mb) deletions and amplifications are detected, based on utilization of test and reference (karyotypically normal) DNA. These samples are whole-genome amplified by DOP-PCR and then differentially labeled with fluorophores via nick translation. Test and reference samples are competitively hybridized to normal metaphase chromosomes. The relative amount of each DNA that binds to a chromosomal locus is indicative of the abundance of that DNA. Thus, if a chromosomal region is amplified, the test DNA will out-compete the reference DNA for binding and fluorescence will indicate amplification. Conversely, if a region is deleted, more reference DNA will bind and fluorescence will indicate a deletion. The following chapter outlines the protocols used for CGH analysis of metaphase chromosomes. These protocols include metaphase chromosome slide preparation, DNA extraction (from blood, cell lines, and microdissected formalin-fixed paraffin-embedded tissue), DOP-PCR, nick translation, in situ hybridization, and fluorescence microscopy and image analysis.
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