Next, p53 exon 8 was amplified using DNA from a group of 48 surgical lung adenocarcinoma samples and s-RT-MELT was used for the screening of unknown mutations via melting curve analysis. Mutations at different positions along exon 8 were present in several of these clinical samples, as indicated by the shift in melting profiles obtained ( Figure 2H ) and subsequently verified via sequencing. In this set of samples, sRT-MELT-sequencing detected a low-level mutation on a colon cancer specimen (CT20) that direct sequencing failed to identify ( Figure 2I ). As with Figure 2F , sequencing of sample CT20 indicated the position of poly-A tail addition to within one base, but the actual nucleotide change was difficult to identify. To exclude the possibility for a false positive, two independent RFLP-based methods were used to verify the presence of the mutation. Thus, since the position of poly-A tail addition was known ( Figure 2I , codon 273 of p53 exon 8) the mismatched primer approach by Behn et al. (42) was used to introduce an MluI restriction site for the wild-type p53 sample but not for the codon 273 mutants. Subsequent restriction with MluI enzyme followed by PCR generated a product with a 14487G4A mutation for the CT20 sample but not for the wild-type sample (Supplementary Figure 1 , Frame A). As an additional verification for the low-level CT20 mutation, we observed that G4A mutation generates a de-novo Nla-III site at the position of the mutation. Accordingly, we applied 'Amplification via Primer-Ligation At The Mutation', a method that we described previously (40, 41) to ligate a primer at the Nla-III-digested site, and preferentially amplified the mutant fragment in a second PCR. The sequenced PCR product identified again the 14487G4A mutation (Supplementary Figure 1, Frame B) . In conclusion, sRT-MELT identified correctly a p53 codon 273 low-level mutation on CT20 that was missed by regular sequencing. This is very significant as p53 exon 8 mutations at codon 273 have been associated with bad prognosis in cancer (43, 44) . Table 2 of Supplementary Data depicts a good agreement between standard Surveyor screening, s-RT-MELT screening and di-deoxy-sequencing, except for the low-level mutation discovered on sample CT20 via s-RT-MELT. s-RT-MELT-sequencing traces for two samples with p53 exons 6 and 7 mutations are also depicted.