Comparative genomic hybridization on BAC arrays.
Alterations in genomic DNA are a key feature of many constitutional disorders and cancer. The discovery of the underlying regions of gene dosage has thus been essential in dissecting complex disease phenotypes and identifying targets for therapeutic intervention and diagnostic testing. The development of array comparative genomic hybridization (aCGH) using bacterial artificial chromosomes (BACs) as hybridization targets has facilitated the discovery and fine mapping of novel genomic alterations allowing rapid identification of target genes. In BAC aCGH, DNA samples are first labeled with fluorescent dyes through a random priming reaction with 100-400 ng of genomic DNA. This probe is then co-hybridized to an array consisting of BAC clones, either tiling the genome (approximately 50 kbp resolution) or spaced at intervals (e.g., 1 Mbp resolution). The resulting arrays are then imaged and the signal at each locus is compared between a reference and test sample to determine the copy number status. The DNA samples to be analyzed may be derived from either fresh, frozen, or formalin-fixed paraffin-embedded material, and sample requirements are currently significantly lower than those for oligonucleotide platforms due to the high probe-binding capacity of BAC clone targets (approximately 150 kbp) compared to oligonucleotides (25-80 bp). In this chapter, we describe in detail the technical procedure required to perform copy number analysis of genomes with BAC aCGH.
Coe
Bradley P
BP
British Columbia Cancer Research Centre, BC, Canada.
Lockwood
William W
WW
Chari
Raj
R
Lam
Wan L
WL
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D055028:Comparative Genomic Hybridization / Q000295:instrumentation / Q000379:methods; D018628:Gene Dosage; D015894:Genome, Human; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods