Comparison of the efficiency of three methods of DNA extraction for deep-sea benthic copepods

Abstract

Meiofauna are small animals that are frequently used as indicators of environmental impacts. Among the meiofauna, Copepoda (Crustacea: Maxillopoda) is one of the most sensitive taxa to environmental changes. It is necessary to extract DNA sequences of copepods to obtain precise estimations of their biodiversity. However, the data presently registered are insufficient not only because of the inefficiencies in DNA extraction and PCR amplification, but also because there is a need for morphological reexamination after sequencing them. In the present study, we compared the efficiencies of the following three methods of DNA extraction for copepods―the Squish Buffer, GeneReleaser^®, and InstaGene methods―with respect to extracted DNA concentrations, morphological characteristics following DNA extraction, in addition to the effect of Rose Bengal staining to PCR amplification. The Squish Buffer method yielded more quantity of DNA than the other methods; however, the morphology of their exoskeleton was better preserved with the InstaGene method. Nested PCR using both the universal and specific primer sets amplified a sufficient amount of DNA for sequencing, even when the extracted DNA concentration was < 10 ng/μL. Thus, DNA extraction by the InstaGene method followed by nested PCR is currently best way to obtain both morphological and genetic information from a single copepod specimen. A comparison of PCR amplification rates indicated that Rose Bengal inhibited amplification, which is consistent with reports in other taxa. The present results should facilitate both morphological and genetic analyses of deep-sea copepods.