Improving the recovery for dissolved eDNA state: A comparative analysis of isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis

Abstract

Environmental DNA consists of multiple states including but not limited to membrane-bound, adsorbed, and dissolved states. Because of their chemical and physical properties, each of these states may have different degradation and transport potential. Essential to the study of eDNA states is being able to isolate them from an environmental sample. Here, we focus on improving the DNA recovery of the dissolved state of eDNA from water. We compared three recovery methods, namely isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis. We evaluated the effectiveness of these methods based on the measured DNA recovery of two different species' DNA of different sizes. DNA recovery was assessed with qPCR. We also checked for the effect of inhibitor removal, and lastly, we estimated the cost of reagents and consumables (not labor). The DNA recovery varied among extraction methods, with isopropanol precipitation yielding the highest recovery (52.47 ± 19.69%), followed by centrifugal dialysis (12.58 ± 7.15%) and magnetic bead extraction (9.92 ± 3.89%). However, isopropanol precipitation's efficacy was influenced by humic acid concentration in the water matrix. The presence of humic acids significantly affected isopropanol precipitation, with higher humic acid concentrations leading to increased DNA recovery. This suggests that humic acids act as co-precipitators during isopropanol precipitation. We observed that longer DNA fragments (chicken) had lower recovery compared to shorter fragments (salmon) across all extraction methods. Magnetic bead extraction effectively removed inhibitors, while centrifugal dialysis and isopropanol extraction required an additional inhibitor removal step. Isopropanol, magnetic bead extraction, and centrifugal dialysis methods have estimated costs of 7.74, 8.53, and 23.62 USD in 2023 respectively. Overall, isopropanol precipitation was the least expensive and most effective with the highest measured recoveries, but it was dependent on humic acid concentration in the environmental sample.