Figure 7.

Amplification of correct target and artefact. Different mixtures of intended correct target and primer-dimer artefact were prepared from isolated products and amplified with PCR using the same primer pair for all reactions [11]. (A) Amplification curves showing that the PCR efficiency, determined from the selection of data points in the window of linearity (blue lines [23]), is the same for the correct target, the mixture and the artefact [12]. The difference in plateau level reflects the different lengths of the PCR products. (B) Graph of the negative first derivative of the melting curves (−dF/dT) reflects the composition of the mixtures that were amplified. The vertical grey lines indicate the melting temperatures of the correct product (90.3 °C) and the primer-dimer artefact (83.2 °C). The presence of the different products in the three reactions illustrates that the observed C_q values (panel A, N_q) are correct (green amplification curve), too low (brown curve) or artificial (red curve). The graphs are based on actual amplification data [11].