Figure 3
From: Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping
Adaptation of SAP for high throughput applications. (A) Diagram illustrating the Amplifluor SNP genotyping assay system. The allele-specific primer each has a unique 5' tail sequence that is identical to the 3' region of one of the Amplifluor SNP Universal Primers (FAM or JOE indicated by green and red arrows respectively). When combined with the common reverse primer, PCR amplification results in the synthesis of the tail sequence complement (thin red line). The Amplifluor® SNPs Universal Primer then anneals specifically to the tail of reverse complement and is elongated by Taq Polymerase. Subsequent PCR cycles unfold the hairpin structure (indicated by filled circle) of the Amplifluor® SNPs Universal Primers, which results in fluorescent signals. (B) A scatter plot showing results of a SAP-based Amplifluor SNP assay. X-axis represents the FAM signal measuring the amplification of WT LUG (+), and the Y-axis indicates the JOE signal that measures lug-16-specific PCR amplification. Two types of controls were used. First, the manufacturer's template controls (GG, GT, TT) utilize FAM/JOE SNP primers and the control templates (GG, TT, and GT), both of which are provided by the manufacturer's kit. Second, the non-target control (NTC) uses water instead of DNA template. Results of three experimental samples (lug-16/lug-16, lug-16/+, and +/+) are shown. DNA template was from known genotype. The experiment has been performed twice with similar results. The result from one such an experiment is shown.