DNA barcoding uses a 650 bp segment of the mitochondrial cytochrome c oxidase I (COI) gene as the basis for an identification system for members of the animal kingdom and some other groups of eukaryotes. PCR amplification of the barcode region is a key step in the analytical chain, but it sometimes fails because of a lack of homology between the standard primer sets and target DNA. Two forward PCR primers were developed following analysis of all known arthropod mitochondrial genome arrangements and sequence alignment of the tRNA-W gene which was usually located within 200 bp upstream of the COI gene. These two primers were combined with a standard reverse primer (LepR1) to produce a cocktail which generated a barcode amplicon from 125 of 141 species that included representatives of 121 different families of Hexapoda. High quality sequences were recovered from 79% of the species including groups, such as scale insects, that invariably fail to amplify with standard primers. The current results show that primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail and provide valuable information to design a primer for problem groups.

• Doo-Sang Park(Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology)
• Soo-Jung Suh(Youngnam Regional Office, National Plant Quarantine Service)
• Hyun-Woo Oh(Industrial Biomaterials Research Center, KRIBB)
• Paul D.N. Hebert(Biodiversity Institute of Ontario, University of Guelph)