title: Analysis of genomic aberrations using comparative genomic hybridization of metaphase chromosomes.
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.
authors.affiliation: Texas Biomedical Research Institute, 7620 N. W. Loop 410 (at Military Drive), 760549, San Antonio, TX, 78245-0549, USA, mcarless@txbiomedgenetics.org.
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Carless
mesh_terms: D002460:Cell Line; D002869:Chromosome Aberrations; D055028:Comparative Genomic Hybridization / Q000379:methods; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008677:Metaphase; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods