Molecular age estimation based on promotor CpG methylation using Methylation Sensitive PCR

Chronological age is a key factor in animal ecology, as many biological traits appear and change over time. Such traits include development, age of reproductive maturity, reproductive success, future reproductive potential, and mortality. Several molecular methods have emerged as potential vehicle for biological age determination. The aim of this experiment is to ascertain if a Methylation Sensitive PCR (MSP) could be developed to screen for methylation at a previously identified site in the GRIA2 gene. Primers were designed by eye for both methylated and unmethylated target CpG’s in the GRIA2 promoter, and MSP conducted with the EpiScope® MSP kit. After optimization, the assay was able to successfully amplify the unmethylated control DNA with an efficiency of 97.6% and R²=99% across a range of 0.3–20 ng input DNA. Comparable results were, however, obtained with the methylated control DNA. Thus, the primers designed for the GRIA2 CpG was able to amplify the selected CpG with great efficiency making MSP a promising method of methylation screening, but primer design to assay a specific site faces many problems and selectivity for methylated vs. unmethylated may not be achievable.