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. 2004 Apr 19;32(7):e63. doi: 10.1093/nar/gnh059

Figure 2.

Figure 2

The quantification of R201H (A) and R201C (B) mutations by using FRET-based DNA hybridization probes. The left panels show the negative first derivative of fluorescence versus temperature of standards (0–50% mutant alleles corresponding to 0–100% mutant cells). (A) Left: the two melting peaks, clearly separated by 5°C, correspond to the perfect match between mutant sequence and mutant fluorescent donor probe (FDP) (melting temperature of ∼65°C) and the single mismatch between wild-type sequence and mutant FDP (melting temperature of ∼60.5°C). There are two peaks because mutant cells have both mutant and wild-type alleles. The one peak in the case of the wild-type sequence was exclusively the lower melting, single mismatch peak. Right: a standard curve was plotted by using pre-mixed ssDNA standards in the 0–50% range of R201H, with the remainder being wild type. (B) Two melting peaks are separated by only ∼1°C (as shown by red and blue bars); thus, they are not easily quantified for the analysis. Standard curves could not be plotted by using DNA standards.