Fluorescent signal scatter in symmetric PCR assays. Replicate sets of homozygous normal (red lines) and heterozygous (blue lines) DNA for the TSD Δ1278 allele of the human HEXA gene were amplified using symmetric PCR. Reactions were monitored using a molecular beacon probe against the normal HEXA allele. The molecular beacon probe is allele-discriminating and recognizes 100% of the alleles in the homozygous normal samples but only 50% of the alleles in heterozygous samples. As a result, the probe fluorescence signals should theoretically be twice as intense in homozygous normal samples compared to heterozygous samples. Each reaction contained 1000 genome-equivalents of genomic DNA. Panel A: Kinetic plots of accumulated amplification products detected by the molecular beacon probe in replicate samples. Signal scattering among replicate samples due to stochastic factors influencing PCR in individual tubes prevents unambiguous identification of homozygous and heterozygous samples based on final fluorescence signal intensity. Panel B: Statistical analysis of data in Panel A. Solid lines represent the average fluorescence intensity values (red: homozygous normal samples; blue: heterozygous samples), error bars corresponds to three-standard deviations of the mean of the fluorescence signals at each amplification cycle which defines the range of fluorescence signals encompassing 99.7% of samples for each genotype. The extensive overlap between the error bars of homozygous normal and heterozygous samples demonstrates that these samples cannot be identified based solely on final fluorescence signal intensity at any amplification cycle.