Effects of water pH and proteinase K treatment on the yield of environmental DNA from water samples
Environmental DNA (eDNA) analysis has recently been applied to the study of aquatic macroorganisms. In most studies, sample water was filtered and the extracted DNA from the residues on the filter used for the following molecular analysis to detect species of interest. This quick, new biomonitoring method has received broad attention, but some unknowns remain, such as the eDNA yield in relation to water quality. Previous studies suggest that eDNA is composed of various forms, such as the free-floating naked form and in organelles and cells. Therefore, the eDNA yield in the filtration and extraction steps might change depending on the composition of eDNA. Especially the filtration efficiency of free-floating DNA would be affected by the electrical effect of water pH. In this study, not only the free-floating naked DNA, but also all DNA fragments released from the organisms and contained in the water were defined as eDNA, including cells and organelles. We examined (1) the effect of water pH on the eDNA yield at filtration and (2) the effect of proteinase K treatment on the extraction efficiency of DNA from filter samples, with consideration of the variety of the eDNA forms in water. In a laboratory experiment using the purified DNA of common carp (Cyprinus carpio carpio) spiked into ultrapure water, the water pH and DNA yield showed a negative relationship within the pH range of 5–9, that is, the DNA yield was higher in acidic conditions, plausibly because of pH-dependent adsorption onto the glass fiber filter at the filtration step. In case the field water contained eDNA derived from the inhabiting common carp and the purified DNA of ayu (Plecoglossus altivelis altivelis) spiked in the sample as an internal standard, adjustment of the pH to 5 prior to filtration did not increase the eDNA yield of common carp, and the spiked ayu DNA was not detected at all. During the DNA extraction step, a standard protocol including proteinase K treatment marked higher DNA yield than that without proteinase K treatment. Overall, the present results indicate successful collection of eDNA using filters without any special attention to the pH of the sample water, and a conventional protocol with proteinase K treatment is appropriate for eDNA recovery.
KeywordsEnvironmental DNA (eDNA) eDNA yield pH Proteinase K treatment
We are grateful to Dr. M. Ushio for his useful comments on the manuscript. This study was funded by the CREST program from the Japan Science and Technology Agency. This study was partly supported by the Environment Research and Technology Development Fund (4RF-1302) of the Ministry of the Environment by Grant-in-Aid for Young Scientists B (26840152) from the Ministry of Education, Culture, Sports, Science, and Technology and by Ryukoku University Technology Fund to HY.
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