Single-egg Comet Assay: a protocol to quantify DNA damage in aquatic dormant stages

Abstract

The comet assay (CA) was originally developed as toxicity test and quantifies DNA integrity from the distribution of DNA across an electric field. Compromised DNA moves across electric fields faster than intact DNA strands, leaving a quantifiable signature that resembles a comet tail. The dimensions of this comet tail reflect relative DNA damage. We optimized the CA protocol for individual dormant propagules (Single-egg Comet Assay or SE-CA) to inform downstream analyses such as DNA sequencing, of the DNA quality contained in natural genetic archives of past populations. As a model we used dormant eggs of the microcrustacean Daphnia. We tested the SE-CA protocol on impact of processing and storage conditions for dormant eggs and used it to assess DNA damage related to aging of eggs retrieved from recently deposited to centuries-old lake sediment. The SE-CA successfully determined the degree of DNA damage in individual eggs frozen in liquid nitrogen, or at -80°C as well as damage caused by bleaching and historical egg age. In conclusion, our protocol provides a cost-effective method of assessing DNA damage in sedimentary propagules such as dormant Daphnia eggs. More generally, the SE-CA can be applied to test DNA integrity in individual propagules prior to genome sequencing or to quantify environmental impacts on natural sedimentary biobanks.