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Thursday, June 27, 2013

Melting Curve Analysis on Real Time PCR

Melting Curve analysis in qPCR is a method of analyzing the dissociation of double stranded DNA during the  heating cycles. The double stranded DNA starts separating out from each other when the temperature is raised, which in-turn causes the change in the intensity of absorbance. melting curve shouldn't be confused with the melting point which denoted by Tm.

Melt curve analysis method can be used to check for any primer dimer formation and other anomalies in the qPCR assay developed.

Melting Temperatures varies from DNA's of different species based on the GC content, more GC content more the temperature required to melt. The reason for this is due to the presence of 3 hydrogen bonds in the GC Pairing as compared to two hydrogen bonds in the AT pairing. So more energy is to break the GC pair.

The temperature dependent dissociation of DNA strands can be measured by using intercalating flourophores like SYBR green, EVA Green or other fluorescent labelled probes. SYBR green binds to the double stranded DNA emits fluorescence, when the temperature is increased DNA denatures / dissociates and becomes single stranded marked by reduction in the fluorescence.

DNA melt curve real time PCR

The graph of the negative first derivative of the melting-curve may make it easier to pin-point the temperature of dissociation (defined as 50% dissociation), by virtue of the peaks thus formed.

One of the other advantage of using the melt curve analysis method is that to find the Single nucleotide Polymerphisms (SNPs).