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Updated: Aug 7, 2026
Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization
Published on: August 6, 2008
Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in
Katleen De Preter1, Filip Pattyn, Geert Berx
1Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium. katleen.depreter@ugent.be
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.
Insights
Researchers developed a new method to quickly identify amplified genes in cancer cells. This approach aids in discovering new oncogenes, improving cancer research and potential treatments.
Area of Science:
- Genomics
- Cancer Biology
- Molecular Oncology
Background:
- Proto-oncogene activation via DNA amplification drives cancer development.
- Traditional gene identification methods are inefficient.
- A novel strategy is needed for rapid cloning of amplified genes.
Purpose of the Study:
- To present a straightforward and efficient strategy for cloning amplified and overexpressed genes.
- To identify novel oncogenes in cancer cells.
Main Methods:
- Utilized PCR-based subtractive cloning to isolate overexpressed cDNA clones.
- Employed custom microarray hybridization with neuroblastoma DNA (IMR-32).
- Combined subtractive cDNA cloning with array comparative genomic hybridization (CGH).
Main Results:
- Successfully detected all previously reported amplified genes in IMR-32 neuroblastoma cells.
- Identified four additional amplified clones, including TEM8, anonymous transcripts, and a fusion transcript.
- Demonstrated amplification sites on chromosome 2p.
Conclusions:
- The combined strategy enables comprehensive amplicon dissection.
- This approach facilitates the identification of previously unknown oncogenes.
- Opens new avenues for cancer research and targeted therapy development.
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