Next-generation melting curve analysis

PCR is not just about amplification.?

If we take a moment to review the various uses of PCR technology, we can discover a whole new world of possibilities and applications for the (now) most popular molecular diagnostic tool.

Melting curve analysis is one of those possibilities.?

In this analysis type, we assess the dissociation characteristics of double-stranded DNA during heating.? This analysis is performed after the PCR is complete and can be done repeatedly (as long as the dye has fluorescence activity) on the amplification products. First, a sequence-specific probe to the target, labeled with a fluorescent dye, is added to the reaction. Then, when the reaction temperature is low, the strand of target DNA is hybridized into the probe, and the fluorescent signal is at its peak, slowly as the temperature is raised, the strands separate and fluorescence is low until none is emitted. This process is the generation of a melting curve.?

A melting peak is generated at the melting temperature - at which 50% of DNA is denatured. More than that, a probe with the same dye can give multiple information - the highest point of the melting peak is unique and depends on the specificity of the probe to the sequence; even one change in the sequence (such as an SNP) can be detected and will appear in a different temp.

Today melting curve analysis is already being used in many fields - ranging from agriculture for genotyping analysis in crops;? to diagnostic medicine for detecting SNPs and mutations and identifying the type of nucleotide substitution in a given sample. Melting curve analysis is also used to discriminate between specific PCR products after a PCR amplification reaction was finished - usually, one melting peak means one particular product of amplification. We can use this reaction characteristic in both research and diagnostic procedures.?

The possibilities for utilization of the proposed above methods are endless and are limited only by the imagination of scientists worldwide.?

One such innovative way to use this type of analysis is to detect dermatophytes in human samples, just like in the kit developed by DYN Diagnostics.

Dermatophytes are hard to diagnose due to the archaic traditional methods in clinical practice. Growing fungi cultures take a long time and have significant false-negative rates, decreasing the test's sensitivity.?

AIDTES software solution is an integral part of DYNs newly developed kit, helping to increase sensitivity and decrease diagnosis time drastically. You can read all about it in our last post about the collaborations between our two companies.

This example showcases AIDTES' flexibility and creativity in utilizing the software for various molecular biology uses.?