( DNA sample is derived from a...)Brilliant Core Reagent Kits for QPCR and QRT-PCR

DNA sample is derived from a homozygous wild-type, heterozygous, or homozygous mutant individual. One way of calling alleles is by comparing Ct values. In a sample containing the wild-type alleles only, the TET signal should be relatively strong and should appear in earlier amplification cycles (Ct = 26), whereas the FAM signal should be at background level (Ct = 40; Figure 3 ). In the case of a heterozygote, both FAM and TET signals should be seen, but the Ct values should be slightly higher than that for the homozygote wild type or mutant because only one allele each (i.e., the wild-type and the mutant allele) is detected. The Ct values for the heterozygote were 27 (TET) and 27 (FAM) (Figure 3).

Another method for calling alleles is to examine the fluorescence at the last annealing stepin this case, at cycle 40. The final fluorescence provides a qualitative readout because components in the reactions might be limiting at the end of PCR. However, when the proper dilutions of target DNA are included in the reaction, the final fluorescence is often related to the amount of the wild-type or mutant allele in the sample (cycle 40; Figure 3) and can be easily extrapolated by the software of the real-time reader (Table 3). In this analysis, the wild-type sample had a TET signal of 2.2 (normalized endpoint fluorescence), the mutant sample had a FAM signal of 1.34, and the heterozygote showed approximately one-half of the TET or the FAM signal.

Table 3
Allelic Discrimination by Endpoint Fluorescence Values

FAM (mutant)

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