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

Discussionlow threshold, we increase the likelihood of also identifying such changes that are presentonly in a minor fraction of the sample.In a diagnostic perspective, all tumor-specific alterations are potentially useful for earlydetection, regardless of their impact on tumor aggressiveness. In this sense, carcinomasamples could also be scored as positive when weakly methylated. However, the currentscoring thresholds provides very conservative results, and ensures that DNA methylation isneither over-estimated in carcinoma samples not under-estimated in normal samples. Thisalso accounts for the resulting sensitivity and specificity measurements.MSP is a highly sensitive method as 1 methylated allele among 1000 alleles is detectable[141].Even though the MSP in itself is qualitative and the scoring of the MSP is visual, the resultsare highly reproducible, which indicates that the band intensities most likely denote theamount of methylated alleles in the lesion. This is verified in paper II, where the MGMTgene has been analyzed in a blinded manner with both quantitative and qualitative MSP. Fullconcordance was achieved, underlining the value of carefully designed assays, well optimizedreactions and thorough scoring of the results.Bisulfite sequencingBisulfite sequencing reads out the methylation status of individual CpG sites in a givensequence and is considered to be the most comprehensive method for studying methylationchanges. Primers are designed to exclude, or limit, the number of CpG sites in order toamplify both methylated and unmethylated DNA in a non-biased manner. A 5-methylcytosinewill be read as a cytosine in the final sequence, whereas an unmethylated cytosinewill be read as a thymine[139].Bisulfite sequencing can be performed either directly on the PCR product or by sequencingclones containing the PCR product. The direct sequencing is fast and simple and will providean average methylation frequency for each of the CpG sites in the entire target sequence. Incontrast, the cloning approach is more work demanding, but will provide exact methylationstatus for each of the CpG sites in the individual clones[144]. The choice of approach46