As a further validation for s-RT-MELT, we utilized the method to identify mutations in additional p53 exons. Figure 3A depicts the chromatographs obtained when a 1:1 mixture of DNA from SW-480 cells (homozygous mutation at p53 14686 C4T exon 9) and from wild-type cells was screened. The real-time PCR reaction was performed at different denaturation temperatures and the products were examined both via dHPLC and via melting curve analysis for comparison. As also observed for p53 exon 8, at 948C denaturation temperature both the Surveyor TM -digested and the undigested PCR products are amplified during real-time PCR ( Figure 3A , curves 1 and 2, mutant and wild-type, respectively). By lowering denaturation temperature to 858C or 848C, a single PCR product is obtained from the mutant while no product, other than primer dimer, is obtained by the wild-type sample ( Figure 3A , curves 3-6). Figure 3B depicts the melting curves obtained following real-time PCR at 858C denaturation temperature for the mutant and wild-type samples. s-RT-MELT was subsequently applied in the same manner to screen for p53 mutations in exons 5-7 from cell lines and surgical colon samples harboring sequencing-identified mutations including a single-base frameshift mutation in exon 7 (listed in Supplementary Table 2 ). The melting curves from mutant and wild-type samples in p53 exons 5-7 are depicted in Figure 3C -E. The data indicate that results similar to those obtained for p53 exon 8 are also obtained for p53 exons 5, 6, 7 and 9.