A simple Dot Blot Assay for population scale screening of DNA methylation

Abstract

The study of epigenetic changes in natural and experimental populations has increased the need to find a cost-effective and high throughput method to analyze multiple samples to effectuate a population-wide screening to study epigenetic changes triggered by biotic or abiotic stress. One of the most studied epigenetic marks is global DNA methylation, its measurement is used as a first step to differentiate methylation between individuals. There is a wide range of methods designed to detect genome-wide 5 methylcytosine (5mC) that differ in sensitivity, price, level of expertise required, but as a general rule, require large amounts of DNA and are relatively expensive. This is a limit for the analysis of 5mC in a large number of individuals as a prerequisite to populationwide testing of methylation markers. In this work, we evaluated a method based on antibody recognition of 5mC to measure the DNA methylation level of individuals of the species Biomphalaria glabrata, the intermediate host of schistosomiases, a neglected tropical disease. We validated the method to complete a large screening in the genome of B. glabrata snails treated with a chemical inhibitor of DNA methylation; however, the method can be applied to any species containing 5mC. The dot blot assay is a suitable method to perform a large-scale screening of global DNA methylation to compare 5mC levels between individuals from different natural or experimental populations. The dot blot method compares favorably with methods with an equivalent sensitivity such as the Enzyme Linked Immunosorbent Assay (ELISA) kit since it requires a smaller amount of DNA (30 ng) is less expensive and allows many more samples to be analyzed.

Beyond the dyad: the role of mother and father in newborns’ global DNA methylation during the first month of life—a pilot study

“…For this reason, this method determines only large variations in DNA methylation, and therefore, it is only suitable for the rough estimation of DNA methylation. However, the dot blot assay is a suitable method to perform a large‐scale screening of global DNA methylation, since it requires a smaller amount of DNA, is less expensive, and allows many more samples to be analyzed in parallel (Luviano et al., 2018).…”

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

“…New DNMTi influence global DNA methylation in vivo over two consecutive generations (multigenerational effect) Using a newly developed population epigenetics screening method that delivered results comparably to ELISA (Supplementary file 2, Figure S4 and S5) but at much lower costs (Luviano et al 2018) we showed that, in the F0 generation, zebularine did not produce a statistically significant difference in 5mC % compared to the control group (W=383, p=0.32). However, 5mC % was significantly different in the groups treated with Flv1 (W=365 p<0.0001) and Flv2 (W=445, p<0.0001) compared to control.…”

“…Finally, the densitometry of the 5mC was analyzed with the software ImageLab5.1. Detailed protocol of this method is found in our preprint (Luviano et al 2018).…”

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

“…Using dot blot method [ 56 ], we found significant differences in the 5mC% between treatments (Kruskal–Wallis test, p < 0.001). In the F0 generation, zebularine did not produce a statistically significant difference in 5mC% compared to the control group (Dunn’s test p > 0.9).…”