Optimized protocol for ctDNA selective capturing on magnetic beads in serum

To tackle the selectivity and specificity challenge, we will employ a technology recently developed by Curie. We have already demonstrated that the use of a microfluidic fluidized bed allows a higher efficiency of DNA capture (from serum) compared to conventional DNA extraction columns. Here we will first functionalize commercial magnetic beads with capture oligos that will permit by hybridization the specific capture ctDNA sequences of interest (KRAS mutated gene will be first considered). Steptavidin beads will be used with biotinylated capture oligos. Different sequence lengths will be investigated to provide the best capture specificity and efficiency. To capture the ctDNA of interest, we will use capture oligo complementary of a non mutated region of the gene of interest, the mutated sequence targeted being subsequently enriched by a first round of ligation.