Figure 2.

In the exon 8 (128 bp) amplicon, a low-abundance mutation in a colorectal tumor sample (CT20) was detected after analyzing COLD-PCR amplicons with HRM (A); however, this mutation was not detected in conventional PCR amplicons. An additional three samples possessed mid- to high-abundance mutations detectable by HRM screening of both conventional and COLD-PCR amplicons. For the exon 8 (128 bp) amplicon, Sanger sequencing analysis (sense strand) of the multiplexed-PCR product for CT20 revealed a G>A mutation in the sequence chromatograms of COLD-PCR (B); however, this mutation was not detected in conventional PCR amplicons. The mutation was confirmed in subsequent analysis of the COLD-PCR-amplified genomic DNA (C); however, the mutation was not observed in either the conventional PCR amplicon or the matched normal sample (D).