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.