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

Discussionthan 96% DHPLC do not stand back in sensitivity for any of these high-throughputmethods. In order to be time efficient and not spend time on method optimization,collaboration with a national diagnostic centre for NF1-related diseases in Rome, Italy, wasinitiated. Here a lab protocol for NF1 analysis was well established, analyzing both sequencealterations and copy number changes[153;154]. The DHPLC primers were generallypositioned approximately 50 to 60 bp away from the intron–exon boundary to allow thedetection of splicing defects while minimizing intronic polymorphisms. Melting temperatureof all fragments was optimized to yield as high sensitivity as possible. In addition, a largenumbers of normal control samples were analyzed and recorded, making it easy todistinguish between mutations and common polymorphisms.Direct sequencingDye terminator sequencing has up until now been considered the golden standard inmutation analysis as it describes any sequence variant with a high accuracy[18]. Still, alimitation with direct sequencing is the detection level. It has been estimated that thismethod can detect and quantify minor sequence variants mutations present in as little as10% of a virus population[155], although other studies claim that this number is as high as30%[18]. The detection level can vary somewhat from sequence to sequence, and althoughwe are able to see base changes at lower cut-offs in a designed sensitivity test using titrationsof known mutations, our experience is that the sensitivity level is approximately 15% forunknown mutations. This means that when running several unknown samples, sequencechanges will be scored when present at this level or higher. Mutations in genes present in asmaller fraction (than 15%) of the sample (e.g. by-stander genes, see page 61-62) will bescored as wild type. In such cases a cloning strategy or another more sensitive method couldbe used, but one might discuss whether it is interesting, in a biologic perspective, to detectmutations that falls below the detection threshold. Such mutations will per definition only bepresent in a few of the tumor cells as discussed on pages 61-62, and are not expected toprovide the tumor with a selective advantage.In addition to the inherent nature of random template selection in PCR assays, the basecallingalgorithm contributes to this relatively poor sensitivity. Although improvements in50