Direct Detection and Quantification of Methylation in Nucleic Acid Sequences Using High-Resolution Melting Analysis

High-resolution melting (HRM) analysis exploits the reduced thermal stability of DNA fragments that contain base mismatches to detect single nucleotide polymorphisms (SNPs). However, the capacity of HIM to reveal other features of DNA chemistry remains unexplored. DNA methylation plays a key role in regulating gene expression and is essential for normal development in many higher organisms. The presence of methylated bases perturbs the double-stranded DNA structure, although its effect on DNA thermal stability is largely unknown. Here, we reveal that methylated DNA has enhanced thermal stability and is sufficiently divergent from nonmethylated DNA to allow detection and quantification by FIRM analysis. This approach reliably distinguishes between sequence-identical DNA differing only in the methylation of one base. The method also provides accurate discrimination between mixes of methylated and nonmethylated DNAs, allowing discrimination between DNA that is 1% and 0% methylated and also between 97.5% and 100% methylated. Thus, the method provides a new means of adjusting thermal optima for DNA hybridization and PCR-based techniques and to empirically measure the impact of DNA methylation marks on the thermostability of regulatory regions. In the longer term, it could enable the development of new techniques to quantify methylated DNA.