Analysis of genomic aberrations using comparative genomic hybridization of metaphase chromosomes.
Comparative genomic hybridization (CGH) allows the global screening of copy number aberrations within a sample. Specifically, large (>20 mb) deletions and amplifications are detected, which are likely to indicate regions harboring tumor suppressor and oncogenes. CGH involves the extraction 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 outcompete 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.
Carless
Melanie
M
Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX, USA.
D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D002877:Chromosomes, Human / Q000235:genetics*; D055028:Comparative Genomic Hybridization / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D017931:DNA Primers / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D021621:Imaging, Three-Dimensional; D017403:In Situ Hybridization; D008677:Metaphase; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA