Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons.
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.
De Preter
Katleen
K
Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, 9000 Gent, Belgium. katleen.depreter@ugent.be
Pattyn
Filip
F
Berx
Geert
G
Strumane
Kristin
K
Menten
Björn
B
Van Roy
Frans
F
De Paepe
Anne
A
Speleman
Frank
F
Vandesompele
Jo
J
D045744:Cell Line, Tumor; D002889:Chromosomes, Human, Pair 2 / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D053487:DEAD-box RNA Helicases; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D000071447:N-Myc Proto-Oncogene Protein; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015513:Oncogene Proteins / Q000235:genetics; D020365:RNA Helicases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D018398:Homeodomain Proteins; C459801:MYCN protein, human; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D000071447:N-Myc Proto-Oncogene Protein; C117339:NBAS protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D012333:RNA, Messenger; D004247:DNA; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases