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

DiscussionFigure 13. Mutation detection using fragment analysis. The left panel is a sample which is wild-type for allfour genes as the electropherograms are identical to the normal control samples. To the right we see a samplewith insertion or deletion in all genes. X-axis – fluorescent intensity, Y-axis – fragment size in base pairs.To date, mutation analyses of close to 200 genes with microsatellites within their codingregion have been published in CRC[90] and 41 of the most prominent genes are included inpaper IV. With a bioinformatic genome wide approach we have identified that more than1000 protein coding genes contain 8 or more mononucleotide repeats in their codingsequence[90]. This implies that genes analyzed so far only represent the tip of the icebergand are not necessarily the ones with most impact on biological or clinical behavior.Multiplex-dependent probe amplificationAs DHPLC fail to detect large genomic rearrangements, another method was included toexamine this. MLPA was first described in 2002 as a novel method to detect copy numberchanges by PCR amplification of several ligated probes[158]. Each MLPA probe consists oftwo oligonucleotides which can be ligated to each other when hybridized adjacent to eachother on a template. All ligated probes have identical 5’ and 3’ sequences so that they can beamplified with universal PCR primers in a multiplex fashion. Only ligated oligonucleotidescan serve as template in a subsequent PCR, eliminating the need for cleaning up unboundprobes[159]. Ligated probes are co-amplified and quantified, and a decrease or increase inthe amount of the amplified probe indicates loss or gain of the exon, respectively.Several controls are included in a MLPA assay. Probes located at different genomic locationsthan the gene of interest are important in order to detect whole gene losses. Control probesscattered around the genome, preferably in regions with known copy number variation can52