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What is the melting curve analysis in qPCR?
Posted June 22, 2020
Answer
Melting curve analysis in qPCR is the assessment of the dissociation characteristics of double-stranded DNA during heating. Researchers often use melting curve analysis to determine the specificity of the qPCR assay, where the intercalating dye is used as reporter who remains associated with the amplicon. A single distinct peak in the plot indicates that the amplified double-stranded DNA products are a single discrete species, i.e., high specificity.
Fluorescent-probe-based qPCR is not compatible with melting curve analysis, because fluorophores are cleaved and released into solution during PCR. However, it is not a big concern for this method because it inherently has a high specificity.
Additional resources
6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*Postollec, F., Falentin, H., Pavan, S., Combrisson, J., & Sohier, D. (2011). Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food microbiology, 28(5), 848-861.Valasek, M. A., & Repa, J. J. (2005). The power of real-time PCR. Advances in physiology education, 29(3), 151-159.