The real-time PCR amplification was performed using Titanium-Taq TM polymerase (BD-Biosciences -Clontech) in a Smart Cycler (Cepheid) real-time PCR machine. For each reaction, we added 0.5 ml polyA-tailed DNA to a final volume of 20 ml with final concentration of 1X Titanium buffer, 0.2 mM each dNTP, 0.1 Â LCGreen (Idaho Technologies), 0.2 mM m13f primer, 0.2 mM oligodT-anchor mix GACCACGCGTATCGATGTCG ACTTTTTTTTTTTTTTTTV [V represents A, C and G each oligodT-anchor concentration is 0.067 mM, as per RACE protocol (35)], 1 Â Titanium TM polymerase (Clontech-BD Biosciences). The thermocycling program was as following: 1 cycle of 948C for 2 min, 25 cycles of 948C for 15 s, 558C for 20 s and 688C for 30 s for reading fluorescence. Temperature titration was performed using different denaturation temperatures, 94-828C to experimentally determine conditions that selectively enable mutation-containing fragments to amplify.