Melting curve analysis

Heat dissociation (PCR product melting curve) analysis at the end of the PCR confirms whether or not a single product is amplified and that no dimers interfere with the reaction. A melting curve analysis begins with heating the PCR product at the end of the PCR reaction. As the PCR product melts and the SYBR® Green is released into the solution, its fluorescence intensity decreases. A negative first derivation curve of the fluorescence intensity curve over temperature produced by the instrument's software clearly indicates the Tm of the PCR product (peak of the -dF/dT curve) and should be quite close to the predicted Tm of the PCR product.

In the example illustrated in Figure 8.5, the PCR product Tm is 87.5°C (curves indicated by +). Complete absence of primer-dimer is rarely achieved in the PCR negative control (curve indicated by -). As seen in this example, 1 out of 3 negative triplicates shows dimers (with a Tm of 78.5°C). The two sets of curves are usually clearly separated with a 10°C shift between Tm of primer-dimers and the specific PCR product. In an experimental negative including a template but no target, dimmers are not usually observed. The degree of an eventual primer-dimer contribution to the overall fluorescent signal of the PCR negative control can also be detected in such a dissociation analysis. Further details of the use of melting curve analysis for allelic discrimination are given in Chapter 9.

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