Two-Temperature LATE-PCR Genotyping. Panel A: Kinetic plots of ResonSense® Cy5 signals collected at 55°C, a temperature at which the probe is allele-discriminating and binds exclusively to the normal allele. Under allele discriminating conditions, replicate homozygous normal samples (red lines) have, on the average, higher fluorescence signals than heterozygous replicates (blue lines) but signal scatter prevents identification of individual genotypes based on this type of kinetic analysis. Panel B: Kinetic plots of ResonSense® Cy5 signals collected at 40°C, a temperature at which the probe becomes mismatch-tolerant and binds both the TSD normal and G269 alleles. Under mismatch-tolerant conditions, homozygous replicates (red lines) and heterozygous replicates (blue lines) exhibit, on the average, very similar fluorescence signals which reflect the total amount of amplification product in each tube. Panel C: Plot of ratio of Cy5 fluorescence signals collected under allele-discriminating (Panel A) and mismatch-tolerant (panel B) conditions. Normalization using the ratio of fluorescent signals collected at temperatures permissive for either allele-specific or mismatch-tolerant probe binding generates characteristic fluorescent signatures that are unique to each genotype. Once established, these signatures do not vary significantly as a function of the extent of the amplification reaction making them suitable for endpoint analysis (red lines, homozygous samples; blue lines, heterozygous samples). Panel D: Statistical analysis of data in Panel C. Red and blue lines correspond to the average fluorescence values of each replicate set of homozygous normal and heterozygous samples, respectively. Error bars correspond to three-standard deviations of the mean which encompasses 99.7% of all possible samples in each replicate set distribution. The absence of overlap between the error bars two cycles after the fluorescent signals ratio is first above background demonstrates that homozygous normal and heterozygous samples can be unambiguously identified by this method with 99.7% certainty.