Abstract
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 (˜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 (˜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.
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Acknowledgments
The authors would like to thank Chad Malloff, Heather Saprunoff, Spencer Watson, and Emilie Vucic for useful discussion. This work was supported by funds from the Canadian Institutes for Health Research, Canadian Breast Cancer Research Alliance, Genome Canada/British Columbia, and National Institute of Dental and Craniofacial Research (NIDCR) grant R01 DE15965.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Coe, B.P., Lockwood, W.W., Chari, R., Lam, W.L. (2009). Comparative Genomic Hybridization on BAC Arrays. In: Pollack, J. (eds) Microarray Analysis of the Physical Genome. Methods in Molecular Biology™, vol 556. Humana Press. https://doi.org/10.1007/978-1-60327-192-9_2
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DOI: https://doi.org/10.1007/978-1-60327-192-9_2
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