Melting curve analysis

Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature. Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. The temperature at which 50% of DNA is denatured is known as the melting temperature. The information gathered can be used to infer the presence and identity of single-nucleotide polymorphisms (SNP). This is because G-C base pairing have 3 hydrogen bonds between them while A-T base pairs have only 2. DNA with a higher G-C content, whether because of its source (G-C contents: E. coli 0.50, M. luteus 0.72, poly d(AT) 0.00) or, as previously mentioned, because of SNPs, will have a higher melting temperature than DNA with a higher A-T content. The information also gives vital clues to a molecule's mode of interaction with DNA. Molecules such as intercalators slot in between base pairs and interact through pi stacking. This has a stabilizing effect on DNA's structure which leads to a raise in its melting temperature. Likewise, increasing salt concentrations helps diffuse negative repulsions between the phosphates in the DNA's backbone. This also leads to a rise in the DNA's melting temperature. Conversely, pH can have a negative effect on DNA's stability which may lead to a lowering of its melting temperature.