Novel genetic and epigenetic alterations in ... - Ous-research.no

up with ExoSAP-IT (USB Corporation, Ohio, USA) and sequencing PCR using BigDye®Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) following the producer’sinstructions. Forward and reverse sequences were analyzed and compared with the genomicreference sequences. For primer details please see supplementary table.Selection of target genesA literature search was performed in order to survey known putative target genes, as alreadyreported.[10] From the 162 genes identified we applied different criteria in order tomaximize the genes with a possible impact on tumorigenesis which in turn will have a higherprobability of determining patient survival.For the initial selection a minimum cut-off mutation frequency of 15% across all tumortypes was chosen following the assessed background mutation level 12-13%.[8;12] To as faras possible ensure that the given mutation frequency was representative, this criterion wascoupled to a requirement of the existence of at least two studies of minimum twenty sampleseach, and a minimum of one hundred samples across all studies. These criteria returned 23genes.In order to include additional molecular markers a second set was defined. This included aminimum observed mutation frequency for the gene of 30%, a minimum of one study oftwenty tumor samples, and involvement in one of the following categories, which were inpart based on Hanahan and Weinberg’s acquired capabilities of cancer: DNA repair, cellsignaling, apoptosis, cell cycle, transcription or angiogenesis.[13]8