MtDNA sequencing from zooplankton after long-term preservation in buffered formalin

Molecular genetic analysis of zooplankton has been slowed by the usual practice of preservation and storage of samples in dilute formalin solutions, which are not always adequately buffered for pH. We report here the determination of DNA sequences for Meganyctiphanes norvegica (Crustacea, Euphausiacea) preserved and stored in buffered formalin for up to 25 years. Specifically designed molecular protocols for DNA extraction and PCR amplification yielded valid sequence data for short (approximately 100-200 bp) regions of the mitochondrial cytochrome b (mtCYB) gene for individual euphausiids. Critical aspects of our approach include: extended extraction and proteinase-K digestion to maximize DNA yield; use of protocols requiring short DNA fragments; design of species-specific PCR primers to minimize risks of contamination by exogenous DNA; and comparison with published DNA sequences for the same gene and species. We conclude that the yield of DNA and the success of subsequent molecular analyses depend primarily on the length of time the tissue has been exposed to formalin and the pH of the solution. Zooplankton samples intended for molecular analysis should preferably be preserved and maintained in ethanol or deep-frozen, but long-term storage in buffered formalin does not preclude some types of molecular genetic analysis.