Use of mtDNA Direct Polymerase Chain Reaction (PCR) Sequencing and PCR−Restriction Fragment Length Polymorphism Methodologies in Species Identification of Canned Tuna

Abstract

Identification of six canned tuna species using DNA-based methodology was studied. DNA was degraded during the canning process of fish muscle: DNA fragment sizes that ranged from <100 up to 200 bp were obtained from canned tuna muscle, whereas DNA sizes for frozen tuna muscle ranged from <100 up to 20 000 bp. Amplification of DNA from canned tuna muscle was carried out using primers flanking a region of cytochrome b gene of 126 bp. Sequences from PCR-amplified DNA of six tuna species were studied for polymorphic sites; seven diagnostic positions were identified in this fragment for the species studied. The suitability of a genetic distance measurement with phylogenetic tree construction method for the identification of canned tuna species using two cytochrome b sequences (299 and 126 bp) was studied. PCR-amplified DNA from canned tuna was also analyzed by using three restriction endonucleases, BsiYI, MboI, and MnlI. The restriction fragments allowed for the identification of the six tuna species studied. Keywords: Canned tuna; species identification; cytochrome b; genetic distance; RFLP−PCR