Evaluation of DNA extraction and handling procedures for PCR-based copepod feeding studies

Abstract

Molecular methods are becoming increasingly common for taxonomic and ecological studies of marine and freshwater plankton. Recently, nucleic acids have been used as target molecules for identification and quantification of prey species in studies of trophic interactions. A critical step in the quantification of mesozooplankton feeding by molecular analysis is the isolation of microalgal DNA from predator guts and in the food environment. It is essential that total genomic DNA extraction provides maximum quantitative yield suitable for downstream analysis. In this study, we compared the efficacy and experimental variability of eight different protocols for total genomic DNA extraction from free-living microalgae and microalgae within the gut of copepods. We also developed and evaluated different sampling procedures for copepods prior to genomic extraction. The optimal protocol was evaluated using real time quantitative polymerase chain reaction (qPCR) and the integrity of the genomic DNA was determined by amplifying PCR targets of increasing size. Considerable variability was observed between purification protocols. Qiagen DNeasy® Blood & Tissue kit was the most efficient of the tested methods for genomic extraction from both free-living microalgae and microalgae inside copepod guts. Furthermore, the appropriate handling of predator copepods prior to genomic extraction was essential for quantitative gut content estimates.