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Interpreting melt curves: An indicator, not a diagnosis

Interpreting melt curves : An indicator, not a diagnosis Researchers often use melt curve analysis to assess whether their intercalating dye PCR/qPCR assays have produced single, specific products. Specificity of amplification is considered to be a greater concern with intercalating dye assays than with probe-based assays because intercalating dyes bind to any double-stranded DNA product and are not sequence specific. With probe based assays, amplicon specific primers gain an additional level of specificity from the probe, which is a third sequence that has to bind within the amplicon of the target sequence in order to generate a fluorescent signal. Melt curve analysis is frequently used as a diagnostic tool for assessing qPCR amplicon length with intercalating dye qPCR assays.

Figure 3. Dissociation curve of CFTR exon 17b amplicon. Change in fluorescence with increasing temperature is measured. As the temperature is increased, the 2 strands of the amplicon separate to form single-stranded DNA, causing the fluorescent intercalating dye to dissociate from the DNA and stop fluorescing. DNA melting is a multi-state process

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