To examine the selectivity of s-RT-MELT, dilutions of mutant to wild-type DNA were performed using DNA from SW-480 cells that harbor a p53 exon 8 14487G4A homozygous mutation. The real-time PCR reaction was again performed at 888C and mutant-to wild-type ratios of $1-10% were distinguished from the wild-type using either dHPLC ( Figure 2D ) or melting curve analysis ( Figure 2E ). In these samples, direct di-deoxy-sequencing could not identify a mutation if the ratio of mutant-towild-type was 5$30-40% (data not shown). On the other hand, sequencing of s-RT-MELT products was possible including the lower dilutions ( Figure 2F ). sRT-MELT sequencing generated traces with poly-A tails depicting the presence and the position of the mutation, although the exact nucleotide change was less clear than the one in exon 5 (i.e. the position AE1 base from the mutation might also be confused to be a mutation). The reason for this AE1 base ambiguity of the exact position of the mutation can be probably understood. The PCR performed following poly-A tail addition contains an equimolar mixture of three reverse primers (3 0 ending in V = G, A or C). Depending on the exact nucleotide at the mutation, the correct primer should in theory be preferred, while the other two primers should not allow efficient polymerase extension due to the mismatched 3 0 -end. However, in practice this 'allele-specific PCR' step occasionally allows 3 0 -mismatched primer extension, enabling more than one version of the primer to amplify over the position of the mutation, or alternatively the incorporation of the poly-A tail may occur AE1 base from the exact position of the mutation. We conclude that in certain cases sRT-MELT indicates the position of the mutation to within 1 base, while in others (e.g. p53 exon 5) it indicates the position 'and' the actual nucleotide change.