Figure 3.

Demonstration of allele-specific primer specificity and examples of Cav-1 mutations identified (P132L and C133R). A: Evaluating the specificity of allele-specific real-time PCR screening methodology, using PCR products of known genetic sequence as the template, ie a WT PCR template (upper panel) or a P132L PCR template (lower panel). Note that primer set 2 preferentially recognizes the WT template, whereas primer set 4 (with the degenerate forward primer) only recognizes the P132L template. Because of a polymorphism at valine 131, primer set 3 did not efficiently recognize either the WT or P132L template. As such, it was not further used. B: Examples of mutations identified using primer set 1 that amplifies the entire Cav-1 gene target region (amino acid 87–156). Upper, wild-type allele P132 (CCA); middle, double mutant example, P132L (CCA→ CTA) and C133R (TGC→ CGC); lower, single mutant example, P132L (CCA→ CTA). Note that in the middle panel, the P132L allele predominates (P132L > WT). Mutations are indicated by the use of blue-green arrows. [Also, several synonymous nucleotide polymorphisms in the Cav-1 gene were identified in our study, eg, the third nucleotide of P132P (CCA → CCA/G) and S136S (AGC→AGT/C). We discussed the chromatogram sequencing results with the Director of the Sequencing Facility at our institution: although the A and G of the P132 were not completely lined up, its location and surrounding nucleotide sequences exclude the possibility of an insertion, and it thus should be considered as a polymorphism.]