With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain.
Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results.
This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance.