SNP genotyping with FRET probes. Optimizing the resolution of heterozygotes

Analysis of single nucleotide polymorphisms by PCR with fluorescence resonance energy transfer (FRET) probes often can produce a result where the melting peak corresponding to perfectly matched sequence (A allele) has a smaller area than the peak corresponding to the allele with a mismatch (B allele). This imbalance can make it difficult to distinguish heterozygous individuals from BB homozygotes. These results suggested that the higher strength in the binding of the perfect match probe to the A allele could cause the selective amplification of the B allele, possibly by interfering with the elongation of the PCR product. In order to optimize the detection of heterozygotes in allelic discrimination assays with FRET probes, we tested several modifications aimed at minimizing the apparent interference of the probes with the amplification process. We observed, in agreement with our hypothesis, that lowering the probe concentration or adding the probes after the amplification step more accurately resolved heterozygotes.