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Characterization of genomically amplified segments using PCR: Optimizing relative‐PCR for reliable and simple gene expression and gene copy analyses
Author(s) -
Jonson Tord,
Mahlamäki Eija H.,
Karhu Ritva,
Gorunova Ludmila,
Johansson Bertil,
Höglund Mattias
Publication year - 2000
Publication title -
genes, chromosomes and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.754
H-Index - 119
eISSN - 1098-2264
pISSN - 1045-2257
DOI - 10.1002/1098-2264(2000)9999:9999<::aid-gcc1026>3.0.co;2-j
Subject(s) - amplicon , biology , gene , genomic dna , gene dosage , gene duplication , gene expression , copy number variation , complementary dna , genetics , polymerase chain reaction , primer (cosmetics) , computational biology , microbiology and biotechnology , genome , chemistry , organic chemistry
Gene amplification is one of the mechanisms for oncogene activation in solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta‐actin ( ACTB ) and L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions. © 2000 Wiley‐Liss, Inc.