Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons
<p>Abstract</p> <p>Background</p> <p>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 posit...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2004-02-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1471-2164/5/11 |
| _version_ | 1811277140820230144 |
|---|---|
| author | De Paepe Anne Van Roy Frans Menten Björn Strumane Kristin Pattyn Filip Berx Geert De Preter Katleen Speleman Frank Vandesompele Jo |
| author_facet | De Paepe Anne Van Roy Frans Menten Björn Strumane Kristin Pattyn Filip Berx Geert De Preter Katleen Speleman Frank Vandesompele Jo |
| author_sort | De Paepe Anne |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>TEM8 </it>gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.</p> <p>Conclusions</p> <p>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.</p> |
| first_indexed | 2024-04-13T00:10:27Z |
| format | Article |
| id | doaj.art-a324ac4e9e434b5db4412aabdc20a519 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-13T00:10:27Z |
| publishDate | 2004-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-a324ac4e9e434b5db4412aabdc20a5192022-12-22T03:11:06ZengBMCBMC Genomics1471-21642004-02-01511110.1186/1471-2164-5-11Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA ampliconsDe Paepe AnneVan Roy FransMenten BjörnStrumane KristinPattyn FilipBerx GeertDe Preter KatleenSpeleman FrankVandesompele Jo<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>TEM8 </it>gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.</p> <p>Conclusions</p> <p>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.</p>http://www.biomedcentral.com/1471-2164/5/11AmplificationOverexpressionOncogeneSSHArray CGHNeuroblastoma |
| spellingShingle | De Paepe Anne Van Roy Frans Menten Björn Strumane Kristin Pattyn Filip Berx Geert De Preter Katleen Speleman Frank Vandesompele Jo Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons BMC Genomics Amplification Overexpression Oncogene SSH Array CGH Neuroblastoma |
| title | Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons |
| title_full | Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons |
| title_fullStr | Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons |
| title_full_unstemmed | Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons |
| title_short | Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons |
| title_sort | combined subtractive cdna cloning and array cgh an efficient approach for identification of overexpressed genes in dna amplicons |
| topic | Amplification Overexpression Oncogene SSH Array CGH Neuroblastoma |
| url | http://www.biomedcentral.com/1471-2164/5/11 |
| work_keys_str_mv | AT depaepeanne combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT vanroyfrans combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT mentenbjorn combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT strumanekristin combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT pattynfilip combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT berxgeert combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT depreterkatleen combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT spelemanfrank combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons AT vandesompelejo combinedsubtractivecdnacloningandarraycghanefficientapproachforidentificationofoverexpressedgenesindnaamplicons |