What is a dissociation curve, and why is it particularly important to run a dissociation curve, following qPCR using SYBR Green chemistry?
Dissociation curves are carried out at the end of a PCR experiment by following a three step procedure First, all of the components are denatured at 95C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve. By analyzing the first derivative of such a curve, one can readily assess the homogeneity of the PCR products, including the presence of primer dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry, due to this reagent’s lack of sequence specificity.
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