Abstract
BackgroundActivation 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.ResultsAs 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.ConclusionsThe 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.
Highlights
Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells
DNA amplification is considered to be a consequence of the intrinsic genomic instability of cancer cells, and it is presumed that overexpression of a single or few amplified genes confers a selective advantage on these homogeneously staining regions (HSR) or dmin bearing clones
We demonstrate that subtractive cDNA cloning followed by Comparative genomic hybridization (CGH) on cDNA microarrays containing the subtracted clones is a powerful strategy for rapid and efficient isolation of amplified genes that are overex
Summary
Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Comparative genomic hybridization (CGH) [4] has been useful for detection of amplified sequences and assignment of the chromosomal position [5] This approach allows whole genome screening for chromosomal imbalances up to 5–10 Mb and gene amplification of sufficiently large amplicons and/or highly overrepresented regions. An important limitation of both methods is the inability to directly identify the overexpressed genes that are targeted by the amplification This limitation was overcome by another variant on the classic CGH approach in which the normal metaphase chromosomes were replaced by a large number of microarrayed cDNA clones [9]. Two limitations of the cDNA array CGH approach are the confined analysis of genes that are present on the array and the analytical challenges in terms of sensitivity by the complexity of the probe and the small sizes of the arrayed target cDNAs (0.5–2 kb) (signal intensities in genomic hybridizations being proportional to the length of the target DNA)
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