Amplicon sequence proportion: A novel method for HRM primer design in DNA methylation analysis among marginalized rural population in Southern Mexico.

DNA methylation is an important modification in the genomes, participating in gene expression or gene repression, as a part of epigenetic studies. This modification can be studied with last-generation sequencing or using PCR coupled with High Resolution Melting (HRM). For this, primers used need to be correctly designed, since the use of specific DNA standards is required, which have specific temperatures displayed in the analyses. We propose and show a method for HRM methylation analysis based on targeted-sequences nucleotide proportion, developed in the Health Laboratory in El Colegio de la Frontera Sur (ECOSUR), Chiapas. We found that when DNA nucleotides in the predicted amplicon have a certain proportion (A-T and G-C), melting curves in the HRM analyses behave differently. Besides, other modifications can be made to primers, such as the number of CpG motifs included within the sequence. DNA nucleotide proportion is shown to be an easy but reliable way of doing primer design when other methods are not available, either because of the lack of resources or the unavailability of sequencing equipment. Additionally, this methodological approach could help reduce time and reagent waste during standardization by improving primer selection efficiency in multi-gene studies.