Figure 1

Illustration of the SAP principle. (A) A step-by-step illustration of the AS primer design for the Arabidopsis seu-1 mutant. The WT (SEU) sequence and the seu-1 mutant sequence are shown on top. The mutated base is underlined. The WT (SEU)-specific primer is first designed based on its complementarity to WT template sequence shown in (1); the MT (seu-1)-specific primer sequence is designed based on its complementarity to the MT template sequence shown in (2). The primer sequence is always from 5' (left) to 3' (right). The penultimate base in the AS primers is indicated by a bracket. Subsequently, the WT primer is paired against the MT template (3) to determine the terminal mismatch (GT). Similarly, MT primer sequence is paired against WT template sequence (4) to determine the terminal mismatch (AC). By referring to Table 1, the GT and AC terminal mismatches identified above both exhibit weak destabilization effect. Thus, the penultimate mismatch should exhibit a strong destabilization. By referring to Table 1, the strongest destabilization mismatch that involves "A" is "GA". Therefore, G is chosen at the penultimate site of both WT and MT AS primers. (B) Four possible annealing scenarios for a hypothetical C to A mutation, which is underlined. Because the terminal mismatches (GA and TC) are strong destabilizing, the penultimate site thus selects a weak destabilizing mismatch (TG), which is indicated within the green rectangle. (1) Proper annealing of a WT primer to the WT template, which will lead to successful PCR amplification. (2) Stable annealing of the MT primer to the MT template, leading to successful PCR amplification. (3) Unstable pairing of the WT primer to the MT template due to two consecutive mismatches. No PCR product is expected. (4) Unstable pairing of the MT primer to the WT template. No PCR amplification is expected.