Research article
Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons
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* Corresponding author: Jo Vandesompele joke.vandesompele@ugent.be
1 Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium
2 Department for Molecular Biomedical Research, Flemish Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, 9052 Zwijnaarde, Belgium
BMC Genomics 2004, 5:11 doi:10.1186/1471-2164-5-11
Published: 3 February 2004Abstract
Background
Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.
Results
As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.
Conclusions
The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.