The sensitivity and specificity in the TP 003 detection of the three mutations obtained in this study were similar to those achieved in the present study. However, this dye-based real-time approach excluded the use of multiplex approach and therefore an internal control cannot be used for more precise quantification. In addition, the multiplexing detection cannot be further explored with this method. The multiplexing ability of our approach may find flexible applications in clinical settings. Current real-time ARMS PCR often have more than 10 primers mixed together in a single reaction, enabling simultaneous detection of multiple mutations but at the cost of specificity restricted to 1�C5%. According to ARMS, an error in primer recruitment would generate a template of the opposite allele, leading to exponential amplification of the error-containing template. In contrast, the specificity of real-time PAP is within 0.1�C0.01%, which is at least 10-fold higher than the ARMS. This is because an error in PAP primer recruitment does not alter the intended template sequence and that the discrimination ability of the PAP primers kept unchanged during the entire amplification process. As shown in this study, increasing the number of primer pairs in one reaction did not impair the analytical performance of either duplex or triplex Bi-PAP. This is due to the fact that Bi-PAP is completely free from primer dimer formation and has negligible mis-priming, and thus increasing primer number would not lead to more non-specific PF 04991532 amplifications. Prospectively, the multiplexing ability of our approach might help build even higher order multiplexing detection. There are two limitations for generally application of realtime Bi-PAP for rare mutation detection. One is the PAP primer preparation, which currently requires multiple reaction and purification steps. In our hand, starting from 10 nmol PAP primer precursors, the amount of PAP primers sufficient for 1,000 reactions can be prepared within 5 working days but with less than 4 h of hands-on time. The other limitation is its fairly moderate increase in detection sensitivity compared with ARMS and DNA sequencing. This makes real-time Bi-PAP not as sensitive as digital PCR in somatic mutation detection. However, since no special instrument is required for real-time Bi-PAP, the cost increase is marginal when compared with a standard real-time PCR while significant equipment and consumable saving can be achieved when compared with digital PCR.