Sequencing of Mitochondrial Genome

We amplify mitochondrial genome of fish in the entirety using a long PCR technique (Miya and Nishida 1999). Usually, four fish-versatile long PCR primers (S-LA-16S-L+H1065-12S and L12321-Leu+S-LA-16S-H) are used to amplify the entire mitochondrial genome in two reactions.

The long-PCR products are diluted with TE buffer for subsequent use as PCR templates. More than 180 fishversatile primers are used in various combinations to amplify contiguous, overlapping segments of the entire mitochondrial

Double-stranded PCR products are subsequently used for direct cycle sequencing with dye-labeled terminators. Primers used were the same as those for PCR.