Multiplexing of E-ice-COLD-PCR Assays for Mutation Detection and Identification

To the Editor: Enhanced- ice -COLD-PCR allows the detection of mutations in a single mutation hotspot down to 0.05% of mutant alleles without prior knowledge of the mutated base, and their identification by subsequent (pyro)sequencing experiments (1). This approach is useful for diagnosis, choice of a personalized treatment, prediction, or the monitoring of treatment response, and has been successfully performed on fresh frozen and formalin-fixed, paraffin-embedded tissue, and circulating cell-free DNA (1, 2). The method is a further development of ice -COLD-PCR (3) and based on the incorporation of locked–nucleic acid nucleotides in the blocker probe complementary to the wild-type (WT)1 sequence (1). During each PCR cycle, denaturation is followed by hybridization at 70 °C, where WT-blocker and mutant-blocker heteroduplexes are formed, mutant-blocker heteroduplexes are denatured at a critical temperature (Tc) higher than 75 °C, and primers are hybridized at the annealing temperature and elongated at 72 °C, leading to the enrichment of mutations underlying the blocker sequence (1). Enhanced- ice -COLD-PCR can enrich all types of mutations, allows stronger mutation enrichment, and is less sensitive to variations of the Tc (±5 °C) compared to full -, fast -, or ice -COLD-PCR, for which a slight variation (0.2 °C) can abolish mutation enrichment (1, 3, 4). Therefore, different full -, fast …