IF positive, sequence the amplified mutated DNA fragment with GC/M13-modified primers we cross-hybridized PCR products and exposed them to Surveyor TM and TdT tailing. The subsequent real-time PCR was run at different denaturation temperatures and the products were examined either via dHPLC or via real-time melting-curve analysis. At the standard denaturation temperature of 948C the mutation-containing sample contains two peaks, corresponding to the anticipated amplification of both Surveyor TM -digested and un-digested fragments (Figure 2A, curve 3) . However, when the PCR denaturation temperature is lowered (e.g. 86-888C) a single PCR product is generated for the mutant sample, while the wild-type sample demonstrates no product (Figure 2A , curves 4-7). In Figure 2B , real-time differential melting curves for the PCR reaction run at 888C are depicted. A peak corresponding to the PCR product from the mutant sample is again clearly evident, which is absent in the wildtype sample. Finally, Figure 2C depicts sequencing of the s-RT-MELT-generated PCR fragment, as well as the direct sequencing from genomic DNA. The G4A mutation is evident in both samples. In the s-RT-MELT product the anticipated addition of the poly-A tail at the 3 0 -position next to the mutation is illustrated.