Melting curve analysis

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Melting curve analysis is an assessment of the dissociation-characteristics of double-stranded DNA during heating. The information gathered can be used to infer the presence and identity of single-nucleotide polymorphisms.

1 Implementation
2 Applications
3 See also
4 External links

[edit] Implementation

Graphs to show the relation between fluorescence and temperature for labeled probe designed for a Wt sequence, homozygous Wt, heterozygous and homozygous mutant situations

The energy required to break the base-base hydrogen bonding between two strands of DNA is dependent on their length, GC content and their complementarity. By heating a reaction-mixture that contains double-stranded DNA sequences and measuring dissociation against temperature, these attributes can be inferred.

The temperature-dependent dissociation between two DNA-strands can be measured using a DNA-intercalating fluorophore such as SYBR green or fluorophore-labelled DNA probes. In the case of SYBR green (which fluoresces 1000-fold more intensely while intercalated in the minor groove of two strands of DNA), the dissociation of the DNA during heating is measurable by the large reduction in fluorescence that results. Alternatively, juxtapositioned probes one featuring a fluorophore and the other, a suitable quencher can be used to determine the complementarity of the probe to the target sequence.

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.

[edit] Applications

The probe-based technique is sensitive enough to detect single-nucleotide polymorphisms and can distinguish between homozygous wildtype, heterozygous and homozygous mutant situations by virtue of the dissociation patterns produced.