Recovery of the mitochondrial COI barcode region in diverse Hexapoda through tRNA-based primers.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
A cocktail of two tRNA-W forward primers coupled with a standard reverse primer amplifies COI for most hexapods, allowing characterization of the standard barcode primer binding region in COI 5' as well as the barcode segment. 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.
Park
Doo-Sang
DS
Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea.
Suh
Soo-Jung
SJ
Oh
Hyun-Woo
HW
Hebert
Paul D N
PD
D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000235:genetics*; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics*; D054629:Genome, Mitochondrial / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000235:genetics*
D017931:DNA Primers; D004272:DNA, Mitochondrial; D012343:RNA, Transfer; D003576:Electron Transport Complex IV