Melting curve analysis

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.

The temperature-dependent dissociation between two DNA-strands can be measured using a DNA-intercalating fluorophore such as SYBR green, EvaGreen or fluorophore-labelled DNA probes.

Many research and clinical examples[5] exist in the literature that show the use of melting curve analysis to obviate or complement sequencing efforts, and thus reduce costs.

Digital High Resolution Melting (dHRM)[6] is also used in conjunction with digital PCR (dPCR) to improve quantitative power by providing additional information on the melting behavior of the amplified DNA, which can help in distinguishing between different genetic variants and in ensuring the accuracy of the quantification.

[7] dHRM is enabled by the use of sensitive DNA-binding dyes and digital PCR instrumentation, which allows for the collection of high-density data points to generate detailed melt profiles.

These profiles can be used to identify even subtle differences in nucleic acid sequences, making dHRM a powerful tool for genotyping, mutation scanning, and methylation analysis[8] dHRM is an advanced molecular technique used for the analysis of genetic variations, such as single nucleotide polymorphisms (SNPs), mutations, and methylations, by monitoring the melting behavior of double-stranded DNA.

The technique has been further advanced by its application on digital microfluidics platforms, which can facilitate the analysis of single-nucleotide polymorphisms (SNPs) with high accuracy and sensitivity.

[10] Additionally, massively parallel dHRM has been developed to enable rapid and absolutely quantitative sequence profiling, which can be particularly useful in clinical and industrial settings where accurate quantification of nucleic acids is critical.