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

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
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.