Report on a ligase-based assay for the selective enrichment of ctDNA targets

In the current task, a ligase-based assay will be investigated for the enrichment of mutant sequences in respect to the normal ones after capturing on magnetic beads. LCR is a DNA template-dependent method performed by a thermostable ligase in the presence of 2 pairs of oligonucleotides. Upon hybridization of the oligos with the template, the ligase connects two adjacent oligos only when perfect matching occurs. This procedure requires cycling of two temperatures for denaturation and ligation and results in exponential accumulation of products without introducing any uncontrolled bias and errors. During LCR protocol optimization the following parameters will be investigated: Oligonucleotides sequence composition and length, number of cycles and temperatures, use of single-base 3’ overhangs, 5’ phosphorylation of oligos, introduction of non-complementary tails and use of cycling conditions near the oligos Tm.