The data in Figures 2A-D and H indicate a lack of substantial PCR amplification at denaturation temperatures 4888C for fragments containing the GC-clamp and a selective amplification of the mutation-containing fragments for several different mutation positions on p53 exon 8. To estimate the influence of the GC-clamp length on PCR efficiency versus temperature and the PCR amplification of fragments generated for mutations lying at different positions along the sequence, a calculation based on the POLAND algorithm (37) was performed. The predicted minimum temperatures for substantial PCR amplification were then plotted versus the experimentally observed values. Three possibilities were simulated, no GC-clamp, 26 nucleotides (nt) GC-clamp and 117-nt GC-clamp. DNA fragments corresponding to mutations at several positions along exon 8 were also simulated and compared to the experimentally observed minimum temperatures for generating a PCR product for three samples that contained mutations at different positions along p53 exon 8 (SW480, CT5 and TL50). The results ( Figure 2G ) indicate agreement to within $1.08C between theoretical prediction and experimental observation. For denaturation temperatures in the region 85-888C in combination with a 26-nt GC-clamp all the available mutations on p53 exon 8 are predicted to result in selective amplification of the mutation-containing fragment and inhibition of the GC-clamp-containing fragment. This prediction is consistent with the experimental results obtained from PCR temperature-titration experiments ( Figure 2G ). The developed calculation algorithm can thus be used to predict the appropriate PCR denaturation temperature for additional PCR fragment/GC-clamp combinations.