An effective procedure for DNA isolation and voucher recovery from millimeter-scale copepods and new primers for the 18S rRNA and cytb genes

Many investigators need to determine whether individuals belong to the same species. DNA-sequence data have helped with this task, but current procedures of DNA isolation from millimeter-scale crustaceans, such as harpacticoid copepods, leave little to no voucher material for morphological analysis, and many procedures yield only enough DNA for a single amplification reaction. We therefore developed a DNA-isolation procedure that yielded essentially intact exoskeletons and sufficient DNA for multiple polymerase chain reactions. DNA-amplification success of our DNA-isolation procedure was relatively insensitive to (1) the length of preservation time from sample collection to DNA isolations and (2) the length of time the DNA was stored at −20°C after isolation. An additional benefit of our procedure is therefore that the DNA isolated is relatively stable. Primers available for the nuclear 18S rRNA gene and the mitochondrial cytochrome oxidase b (cytb) gene are known not to work for many harpacticoids. We therefore designed primers that would amplify and sequence an ~750-base-pair fragment of the 18S rRNA gene and others that would amplify and sequence an ~450-base-pair fragment of the cytb gene. Both primer sets worked for at least 12 harpacticoid families. •We report a procedure for DNA isolation and exoskeleton recovery for single copepods.•This procedure yielded enough DNA for tens of polymerase chain reactions.•This procedure isolated DNA from specimens preserved for up to 52 mo after collection.•This procedure amplified DNA for up to 32mo after DNA isolation.•We report new primers for the nuclear 18S rRNA and mitochondrial cytb genes.