Identification of imbalance allele ratios using the Two-Temperature LATE-PCR endpoint assay. Replicate samples (n = 16) containing 1000 genome-equivalents of heterozygous TSD G269 DNA (1:1 C to G allele ratio) or a DNA mixture containing a 2:1 ratio of the same two alleles (see Materials and Methods) were analyzed by Two-Temperature LATE-PCR using primer/probe pairs for the TSD G269 polymorphism. Fluorescence signals were collected prior to LATE-PCR amplification at 70°C and after LATE-PCR amplification at 52°C and 40°C to calculate the normalized endpoint fluorescence ratios (see Materials and Methods). Blue dots, heterozygous (1:1 C-toG allele ratio) fluorescence ratios; green dots, DNA mixture (2:1 C-to-G allele ratio). The black boxes represent the boundary of three-standard deviations on either side of the mean of replicate control samples signals for each genotype and statistically define the 99.7% confidence interval for identification of each genotype.