Figure 3.

In the exon 9 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 detectable in conventional PCR amplicons. An additional six samples possessed mid- to high-abundance SNPs detectable by HRM screening of both conventional and COLD-PCR amplicons. For the exon 9 amplicon, Sanger sequencing analysis of the multiplexed-PCR product for CT20 revealed a C>T mutation in the sequence chromatograms of COLD-PCR (B, anti-sense strand sequencing presented); however, this mutation was not detectable in conventional PCR amplicons or the matched normal sample (D). The mutation was confirmed in subsequent analysis of the COLD-PCR amplified genomic DNA (C).